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1. Choose Print Preview from the File menu 2 A window will appear showing the print layout prior to printing Print Setup This function allows the user to setup the printer layout according to their own specifications 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Artisan Technology Group Quality Instrumentation Chapter 4 ProGest Method Editor Customization Choose Print Setup from the File menu The Print Setup window appears Choose the appropriate settings Select OK when finished Edit This command allows the user access to editing functions such as insert step delete step append step and edit vial contents names Delete Step E This command allows the user to delete a selected step in the opened protocol T Select Delete Step from the Edit menu Note This step will only be operable if a step is selected high lighted in an opened protocol in the Method Editor window A warning message will ask the user if they are sure they want to delete this step Click Yes to delete the step Investigator ProGest 3 28 01 61 Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Insert Step This command allows the user to insert a step in the opened protocol 1 Select Insert Step from the Edit menu The New Method Step window appears Hew met
2. Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization List of Steps Below is a list of protocol steps available with the ProGest Depending upon the step selected one or more windows are required to enter the relevant information needed for the completion of the step parameters These steps may be added by either Insert Step or Append Step as discussed previously For more information about each step refer to the page listed Aliquot to each sample well takes a quantity of liquid from a vial or reservoir and distributes it to the active wells on the sample plate Refer to Aliquot to each sample well on page 70 for more information Heater Control sets the temperature to either low 37 C high 60 C or Off Refer to Heater Control on page 72 for more information Incubate purge aliquot delay directs the system to purge one liquid from a sample tube and replace it with another The delay is provided to allow time for a reac tion to take place prior to the instrument moving on to the next step Refer to Incubate purge aliquot delay on page 72 for more information Move the sample rack directs the system to move the sample rack to a designated location Refer to Move the sample rack on page 76 for more information Pipette from one vial to another directs the system to transfer liquid from one vial to another This step can also be used to stir
3. 2 If particular positions on the 96 well plate are desired select the desired quadrant by touching the screen in that quadrant Note that wells are processed in pairs due to the twin probe configu ration A window will appear showing the sam ple positions in that quadrant 3 Touch the desired positions to select them If selected they will appear white The user may choose to Select All which changes the color of all the wells in that quadrant to white Investigator ProGest 3 28 01 27 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation 28 B 1 1 a a S es mmm Ses The user may select to Clear All or clear each two sample position by touching the appropriate sample block The sample pair will go dark indi cating that it is no longer selected When you are finished selecting the appropriate sample positions select Done to return to the previous window It is not necessary to access all quadrants The previous settings for a quadrant will be maintained or you may repeat steps 2 through 4 to select wells in another quadrant When you are finished selecting the appropriate sample positions select Done to return to the previous window The window will appear with check marks in the quadrants that has been indi vidually altered The quadrants will appear as White all wells selected
4. or 1 734 975 4800 FAX 1 734 975 4808 Investigator ProGest 3 28 01 111 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 6 Troubleshooting For rest of world please contact your local distributor or contact Genomic Solutions by E mail help genomicsolutions com If you are in Japan call Tel 81 3 3280 0990 FAX 81 3 3280 099 On the internet http www genomicsolutions com or contact us at Genomic Solutions Inc 4355 Varsity Drive Suite E Ann Arbor MI 48108 or at our Lansing Manufacturing facility Genomic Solutions Inc 3120 Sovereign Drive Suite A Lansing MI 48911 112 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Appendix A Trypsin Digestion Kit Appendix A Trypsin Digestion Kit The Genomic Solutions ProGest Trypsin Digestion kit is designed for the tryptic digestion of protein gel slices or spots using the Investigator ProGest Protein Digestion Station The reagents contained in this kit will process up to 96 samples using any of the three protocols provided with the ProGest In addition to these contents HPLC grade acetonitrile 100ml and 18 megohm cm water are required The following instructions describe reagent preparation and placement in the ProGest sample work area Safety This kit is for use by qualified laboratory st
5. 00000 5 97 Check Hester 1o boxe x y ensis EDU EXC RTI EE 98 Network Address 2 222422 e 4 REL iva ERR RS 99 er un Cnm 100 Shut down long periods 0000005 100 6 Troubleshooting eens 101 Machine Test Method 4 101 Troubleshooting Tips ois sux rrr RE ra 104 Technical Assistance 2 le hn 111 Appendix A Trypsin Digestion Kit A 113 SAEY Seta ood eiu aded da DEAE Ru quA A 113 Reagent Preparation lslseees esee A 113 Prepare Solvents C and D A 114 Prepare DTT Reagent A A 114 Prepare Iodoacetamide Reagent B A 114 Prepare Trypsin Enzyme C A 114 Prepare Formic Acid Solvent E A 115 Prepare Water Solvent F A 115 Vial Placement 445504464 b n RR xx x A 115 Running the Protocol 004 A 116 iv 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Contents Appendix B ProGest Validation Kit B 117 OMe aure oaa d 495 4094 cm e eyed ums B 117 nstructions ilex cana ek a acce Ka cd Roe E e B 118 Preparing Peptides for MALDI TOF B 119 MALDI TOF Results 0 0 B 119 Investigator ProGest 3 28 01 V Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Con
6. A rtisan Artisan Technology Group is your source for quality TecmoogyGroup new and certified used pre owned equipment FAST SHIPPING AND SERVICE CENTER REPAIRS WE BUY USED EQUIPMENT DELIVERY Experienced engineers and technicians on staff Sell your excess underutilized and idle used equipment TENS OF THOUSANDS OF at our full service in house repair center We also offer credit for buy backs and trade ins IN STOCK ITEMS www artisantg com WeBuyEquipment EQUIPMENT DEMOS HUNDREDS OF InstraV ea REMOTE INSPECTION LOOKING FOR MORE INFORMATION MANUFACTURERS Remotely inspect equipment before purchasing with Visit us on the web at www artisantg com 7 for more our interactive website at www instraview com 7 information on price quotations drivers technical LEASING MONTHLY specifications manuals and documentation RENTALS ITAR CERTIFIED yop aed Contact us 888 88 SOURCE sales artisantg com www artisantg com ontents Warnings amp Safety Precautions l Safety Warning sozeccacbiixXd e kERERAR AEG Eq ep aenw as I Safety amp Operating Symbols 0 0 II Installation HP II Power Requirements siesoseswt a e RR RER EA III Specifications usi dace y Seer eee eS ce DAN ARA MeRES III Instrument Maintenance 0 00 seen IV Letter of Conformity CE 004 VI 1 Introduction siii tte ede eR e aea e EE Ra wae 1 System Components 0 0 e
7. Enter the reagent source of the aliquot to be added from the drop down menu list Enter the reagent destination from the drop down menu list Note A useful feature to mix the contents of a position is by specifying the source and destination as the same Enter the aspiration volume in pl Enter the dispense volume in ul Dilutor Enter an aspiration and dispense speed in ml mm e Select an airgap volume in ul if desired Select an equalization time in seconds Step Sequence specified air gap is aspirated needles move into source vial 78 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization specified volume of liquid is aspirated needles move to destination vial specified volume of liquid is dispensed if aspiration volume is greater than the dis pense volume the excess is discarded to waste Rinse needles This step directs the system to wash the needles to avoid contamination of the samples or reagents Refer to Rinse on page 68 for more information Start a timer This step directs the system to start a selected timer but allows the method to continue running You may have up to four timers running simultaneously in a method Once a timer has been started it steadily counts down to zero regardless of what else the ProGest is doing For exam
8. Low will be displayed and the heater will start to heat the tray to 37 C The dis play on the heater controller on the front of the ProGest will show two temperatures The top temperature will be the actual sensed tempera ture of the tray and the bottom reading will be the demand temperature Note All temperature controls should be made through the software 98 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 5 Maintenance The temperature controller on the front of the ProGest should never be adjusted 4 Ifthe Heater button in the Setup window is pressed again then high will be displayed in the setup window and the heater will start to heat the tray to 60 C The top temperature reading on the temperature controller front of ProGest will be the actual sensed temperature and the reading below will be the demand temperature 5 Press Heater again and the heater will be turned off You may also select Done to turn the heater off 6 If there is a problem with obtaining either heater temperature contact Genomic Solutions Techni cal Service for assistance Network Address If multiple ProGest or ProMS units are to be networked together a unique network address must be selected for each instrument to enable communication between each specific unit and the network To access this Network Address 1 Select Plate
9. using a clean pipette tip cut so that the internal diameter of the tip gives a gel plug of up to 2mm in diameter or by using a clean scalpel blade This process can be readily automated using the Flexys Protein Picker in which protein features of interest are cored and placed into the red colored Reaction plate Investigator ProGest 3 28 01 19 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation Loading the reaction wells Reaction Block TEE LINES riot d ph ee dh aa dg Mi kh di ni m di i Reaction Plate Cover To load the reaction plate containing the gel pieces 1 Remove the Reaction Plate Cover by removing the four corner screws that hold the Reaction Plate Cover onto the Reaction Block 2 Place the red colored Reaction plate containing the samples into the Reaction Block with the A1 well located at the back on the left 3 Place the plastic film on top of the sample tray using gloves to avoid contamination 20 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation 4 Replace the Reaction Plate Cover with the four corner screws finger tighten by quarter turns on the diagonal nuts to ensure an even seal The plate has 96 O rings on the underside which seal the film to the red plate These screws need to be tight to ensure
10. 1 Prepare reagents for the ProGest using the ProGest Trypsin Digestion kit Follow the instructions in the kit for reagent preparation and placement on the ProGest 2 Remove the pink 96 well plate from the ProGest Validation kit plastic ziplock bag Carefully tap the pink plate on a flat surface to ensure that the blue protein gel plugs are all on the bottom of the wells 3 Remove the plate cover carefully 4 Follow the instructions starting with Loading the reaction wells on page 20 to run the ProGest using the Default Trypsin Digestion protocol 5 When the run has completed and the peptides are extracted into the blue plate place the blue plate in a vacuum microcentrifuge fitted with a 96 well microplate rotor and evaporate to dryness B 118 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Appendix B ProGest Validation Kit Preparing Peptides for MALDI TOF T Remove the small vials of matrix and matrix solution from the small ziplock bag labeled ProMS Matrix Add the matrix solution to the vial containing the a cyano hydroxy cinnamic acid matrix Shake the vial to mix the contents thoroughly the matrix will not completely dissolve in the solution If using the ProMS workstation resuspend the peptides in the blue plate in 100ul of 10 Formic Acid Place the tube of prepared matrix solution in the ProMS workstatio
11. Configuration from the keypad 2 Select Setup from the keypad 3 Scroll up by selecting the Address button or down using the Address button until the desired address number is reached 4 Select Done when finished Investigator ProGest 3 28 01 99 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 5 Maintenance Cleaning The protective cover should be washed with warm dis tilled water Do not use Methanol or any other solvent The tray and the surface of the instrument can be cleaned with a cloth dipped in soapy water The needle guide and needle should be washed with methanol using a spray bottle Shut down long periods Some of the chemicals used are hazardous material which must be disposed of properly Check with your safety officer and follow the regulations of your state or institute For long periods of shut down time we recommend the following procedure 1 Switch off the system and remove the power cables 2 Empty and clean dilutor reservoir and waste bot tle 3 Empty and clean work area 100 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 6 Troubleshooting Troubleshooting The ProGest is a sturdy instrument assembled from reli able parts However as in every instrument there are possibilities of unexpected mal
12. Dim March 15550 VI 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 1 Introduction The Investigator ProGest Protein Digestion Station ProGest is an automated instrument for the enzymatic digestion of proteins contained in pieces of gel after elec trophoretic separation The ProGest performs enzymatic digestion on up to 96 samples simultaneously It auto mates the following steps Washing of the gel slices with various buffers Reduction of disulfide bonds at 60 C Alkylation of cystine residues with iodoaceta mide Removal of excess reagents Equilibration with digest buffer Addition of digestion enzyme optional up to 4 enzymes Incubation at 37 C for digestion Elution of peptides from the gel matrix Investigator ProGest 3 28 01 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 1 Introduction System Components The major components of the system are Pipetting robot Dual syringe dilutor unit Work area with heatable reaction block sol vents and reagent rack collection rack Built in Touchpad and Liquid Crystal Display single board computer plus disk drive 3 5 HD disks only and operating software Protective cover 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 S
13. Method Editor Customization Wait for the operator This step directs the system to pause and wait for the operator A message is displayed for the operator and processing will continue when the operator presses the OK button on the ProGest screen Note Make sure the sample tray is not moved during this time as the tray is not homed after this pause Investigator ProGest 3 28 01 81 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Network This command allows the user to communicate with one or more ProGest Investigator ProMS or other Genomic Solutions instrument via a cable connected to one of your PC s serial ports Settings This command allows the user to enable or disable the network feature and to select the particular port on your PC to which the network is connected 1 Select Settings from the Network menu The Net work Settings window appears Select to network systems by checking the Enable Genomic Solutions network checkbox Select the correct COM port to be used COM 1 to COM 9 normally COM 1 or 2 This screen also shows the maximum number of network addresses it queries one for each instru ment networked Note The system always defaults to 8 When you are finished select OK to close the win dow Network Status The network view shows a short indication of the status of ea
14. N mbr Description 10 Wash with Bicarbonate Source 25mM Bicarbonate Vol 100yl Hold Time 10 minutes 0 seconds 11 Purging ABC Source Acetonitrile Vol Oul Hold Time 0 seconds 12 Washing with CH3CN Source Acetonitrile Vol 50ul Hold Time 0 seconds 13 Start Timer Starting Timer 1 Time 15 minutes 0 seconds 14 Neutralizing with ABC Source 50mM Bicarbonate Vol 50ul 15 Reaction Time Waiting for Timer 1 16 Shrinking gel Source Acetonitrile Vol 100u1 Hold Time 10 minutes 0 seconds 17 Set Heaters to High Heater HIGH 18 Reduction with DTT Source DTT Vol 30u1 Hold Time 10 minutes 0 seconds 19 Turn off Heaters Heater OFF Investigator ProGest 3 28 01 43 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols Step Number Description 20 Reduction cooling Delay 20 minutes 0 seconds 21 Alkylation Source Iodoacetamide Vol 30u1 Hold Time 15 minutes 0 seconds 22 Washing with ABC Source 50mM Bicarbonate Vol 40ul Hold Time 10 minutes 0 seconds 23 Washing with CH3CN Source Acetonitrile Vol 50u1 Hold Time 15 minutes 0 seconds 24 Shrinking with CH4CN Source Acetonitrile Vol 50u1 Hold Time 15 minutes 0 seconds 25 Purge wells Source Acetonitrile Vol Oul Hold Time 10 minutes 0 seconds 26 Prepare enzyme Source 25mM Bicarbonate Vol 950u1 Destination Trypsin Vol 900u1 27 Mixing enzyme So
15. ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 842 51 1 834 472 799 894 963 539 016 44 1219 59 1210 68 Bihl 1737 89 1425 64 495 71 1350 44 hi 1676 55 1917 93 1894 09 Appendix B ProGest Validation Kit 2242 17 2211 2 A 9 13 wl i l hi i DT NENNEN Search Results 1 7 29 matches 24 55561 6 Da pI 8 35 Acc f P00366 BOVIN GLUTAMATE DEHYDROGENASE EC 1 4 1 3 GDH m z submitted 956 5120 963 5393 1000 4631 1016 4431 1210 6772 1425 6392 1737 8936 MH Delta matched ppm 956 5205 8 8885 963 5264 13 4032 1000 4562 6 8996 1016 4511 7 8822 1210 6948 14 5273 1425 6286 7 4293 1737 8924 0 7087 Fig 4 start 397 471 12 12 296 424 end 403 478 19 19 306 439 Peptide Sequence R LTFKYER D K YNLGLDLR T K MVEGFFDR G K MVEGFFDR G 1Met ox K LOHGTILGFPK A R DDGSWEVIEGYR A K HGGTIPIVPTAEFQDR I Mass spectrum of the peptide digest from sample 4 obtained using the ProGest Validation kit Investigator ProGest 3 28 01 B 123 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Appendix B ProGest Validation Kit 842 51 927 497 Ah font 1163 62 1097 55 1567 75 1479 8 1439 82 1305 71 18 128
16. SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization jahaaa Dii Page Firm nie Dario eT IT LECT om a This screen refers to washing the needles to avoid con tamination of the samples or reagents This step per forms a two stage rinse First with the needles above the rinse port so that the inside of the needles is washed then with the needles inside the rinse port so that the outside of the needles is washed The default for both washes is 1000u1 Source This option in any of the pipetting or dispensing steps opens a sub menu or list of vial contents names Investigator ProGest 3 28 01 69 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization sed Diis Vial positions are referenced to the vial contents names These positions are factory programmed as are other crit ical features and can not be accessed by the user For more information about the positions associated with each vial content name refer to Appendix A Trypsin Digestion Kit on page 113 Aliquot to each sample well This step takes a quantity of liquid from a vial or the res ervoir and dispenses it to each of the active wells in the sample plate The desired amount of reagent along with the reagent name must be entered An aspiration speed dispense speed airgap volume if liquid is drawn from a vial rather than t
17. are removed between each step by a series of dehydration and rehydration steps as described above After reduction and alkylation trypsin stored in acidic buffer to prevent autodigestion is activated by the ProGest by diluting the enzyme with ammonium bicar bonate buffer pH 8 Activated enzyme in ammonium bicarbonate buffer is added to each gel sample and is incubated at 37 C for approximately 1 5 hours depend ing on the methodology selected Each well in the red colored Reaction plate receives a further small aliquot of water to replace any solution lost through evaporation and is incubated at 37 C for a further 1 5 hours When the trypsin is added the Reaction Block containing the red colored Reaction plate is moved from above the waste position to the collection position Here the result ing peptides are extracted from the gel by a series of Investigator ProGest 3 28 01 9 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 1 Introduction washes with ammonium bicarbonate buffer formic acid and acetonitrile an acidic basic and organic elution Typically the peptides are immediately dried by freeze drying or in a vacuum centrifuge The peptide is resus pended in an appropriate buffer ready for analysis by mass spectrometry LC or CE or Edman degradation For analysis by MALDI TOF mass spectrometry using the Investigator ProMS system the peptides can be take
18. clicking on the appropriate part of the plate When you are finished select OK The new plate settings will be sent to the ProGest and stored within the ProGest Method Transfer To send a method from the Method Editor to the ProGest 86 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Click the right mouse button on the device in the network view A window will appear Select Send Method The method currently loaded open in the editor will be sent to the ProGest It will not be started automatically Investigator ProGest 3 28 01 87 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization 88 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Artisan Technology Group Quality Instrumentation Chapter 5 Maintenance 5 Maintenance The ProGest has been designed to reduce maintenance to a minimum However some maintenance will eventually be required Only maintenance operations described in this manual should be carried out by the user Other maintenance operations not described here should be referred to Genomic Solutions Technical Service S Warning Before beginning any maintenance operatio
19. directly to the ProGest It also allows remote control start stop sample selection of the ProGest instrument In addition the Method Editor lets you view all systems networked to your PC by means of the main Method Editor screen which shows a set of pan Investigator ProGest 3 28 01 53 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization els one for each device on the network Each panel con tains a summary of the type of instrument and its current status You are able to control each instrument by click ing on its panel in the network view Loading Software 1 Download the Method Editor software from the Genomic Solutions website directly into your com puter 2 Follow the prompts in the installation program 3 After installation or when you restart Windows the Method Editor will be available via the Start menu If you are not familiar with Windows refer to the Windows Help for guidance 4 Open the program by selecting and double clicking on the icon The Method Editor window appears Method Editor Window The main window of the Method Editor is divided into four sections 54 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Menu amp Tool Bar Step List S
20. ee eee eee eee 2 System Software oacavdiccGe debe ES dDCREN ted ERN 3 System Hardware 2 26222ke a RE Rea RE aS 4 System Overview i icosciie es aste ra Rx RR T adn 5 Method MUR a e ak e a ERE Sia 7 Protein Identification 0 0 0 7 Principle of Operation ProGest 8 Protein Digestion Methodology performed by the ProGest Default Method 0 9 General Safety Procedures iso sone a 9 tua 10 About the User s Manual lesen 12 2 Operallon i542 ucbrad Esau R ERE aes 15 Preparing the system 22222 cb Res pXES REY kaDEUS 15 Empty waste bottle 0 0 0 00000 15 Preparing reagents 222 2454234s ernseesecevaess 16 Investigator ProGest 3 28 01 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Contents Loading the work area 4 2222 oer Rn 17 Isolating protein samples lesse 19 Loading the reaction wells 000 20 Preparing the Reaction Plate 22 Removing the Reaction Block 23 Load the collection rack 14d sant 24 Switching the system on 008 24 Sete FP Po c m 25 Method Selection 0 0 0 eee ee eee 26 Plate Configuration 432 e de PERSE Poe ee ERES 27 PAMIDS n04 eeu rude E ERES Ed eee seus 29 Start DIBSSIGE iid ci Daccaa dece ed tees Sueeneeesd 30 Interrupting Aborting the program 31 Handlin
21. electrician should be contacted Both the ProGest and its accessories will be pre set at the fac tory for the correct voltage for its destination The volt age switch setting for the ProGest is internal and should not be accessed except by Genomic Solutions personnel used by the ProGest could lead to a potential shock haz Warning Failure to provide a well grounded outlet QD ard Specifications General Dimensions 41cmW x 61cmD x 67 1cmH 15 8 W x 24 D x 26 4 H Weight 54 8kg 121 Ibs Electrical Mains input 220 240V ac or 100 120V ac e 50 60 Hz 150 VA max Investigator ProGest 3 28 01 Hl Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Safety Precautions Environmental The ProGest is intended for safe operation under the fol lowing conditions Indoor use Up to an altitude of 2000m Between the temperatures of 5 C and 40 C Maximum relative humidity of 80 for temperatures up to 31 C decreasing linearly to 50 relative humid ity at 40 C Where mains supply voltage fluctuations do not exceed 10 of the nominal voltage Environment should be dust and vibration free Computer not supplied 2MB of internal disk storage Color monitor minimum screen resolution 800x600 Windows 95 98 or Windows NT 4 0 or later RS232 port for cable connections to ProGest Instrument Maintenance Clean the ProGest ex
22. in the 96 well microtiter plate are two small holes The holes are sufficiently small so that the solution does not leak from the well The solution is only expelled when the outer probe forms a seal with the film and nitrogen gas pushes the fluid through the small holes into the blue colored Collection plate mounted on the Collection rack The microtiter plate well therefore serves as a flow through reactor and is the key element in a series of reactions performed by the ProGest 8 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 1 Introduction Protein Digestion Methodology performed by the ProGest Default Method The gel pieces contain high concentrations of buffers and detergents that are typically used in all standard separa tions performed by 1D and 2D electrophoresis These must be removed from the gel so that effective digestion of protein samples can be performed with trypsin This is achieved by dehydrating the gel with several successive washes with acetonitrile followed by rehydration with bicarbonate buffer Natural proteins often contain disulfide bridges that maintain the secondary structure of the protein and would otherwise interfere with the digestion of the protein by trypsin The ProGest reduces these by incubating the gel pieces with DTT at 60 C and alkylates the free sulfhy dryl groups with iodoacetamide Excess reagents
23. next step in the opened protocol Edit Vial Contents Names This command allows the user to edit the descriptions of the vials contents and customize the name of each reagent solvent and enzyme solution Investigator ProGest 3 28 01 63 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Note Each of the vial names is associated with a specific location on the ProGest for ease of use when method editing Refer to Loading the work area on page 17 for more information To enter or change a vial contents description 1 Select Edit Vial Contents Names from the Edit menu The Vial Contents Names window appears Viel content namas Aeserene Sokeri i Sokeri B Soki L Solvers D Solent E Sokeri F Reagent A Reagent Eran X Erzyme E Enzymes C Ergma D Shp 2 Select highlight the desired vial name 3 Double click on the name or select Edit to bring up the Vial Contents Description window Type in the name or description of the vial contents Select OK when finished The new description should appear in the Vial Contents Names window and in any subsequent steps that select it Note Any steps that selected that source or reagent prior to changing the contents description will not be updated until that step is selected again 64 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation
24. nut at the top of the syringe 1 Mount the new syringe Screw the threaded end of the the syringe body into the valve Lower the piston so that it comes it contact with the piston holding screw Note For new syringes the piston may be difficult to lower Attach the syringe press OK and while the drive is slowly being raised align the syringe and base until it homes properly Tighten the mounting screw at the bottom of the piston operating rod 3 28 01 93 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 5 Maintenance 10 Press OK to bring the syringe back to home posi tion 11 Press Done until you get back to the main menu 12 Replace the plexiglass cover making sure to re position the feed line at the right side of the win dow to prevent line damage Probe Adjustment If the reaction tray is accidentally moved during a run a probe crash may be possible As a result the inner black probes may be pushed up inside the outer stainless steel ones Before the run can be re started the inner probes must be adjusted to the correct length To reset the inner probes the following procedure must be observed Turn the ProGest off at the main switch front of instrument Remove the probe assembly from the robot arm by unscrewing the black thumbscrew and lifting the probe assembly clear Undo the brown locking screw that holds the probes in the
25. ple you start timer 1 for 10 minutes the ProGest pro ceeds with the run steps which takes 5 minutes then waits for timer 1 to count down to zero system waits for a further 5 minutes this step should be used in conjunc tion with a Wait for a timer step HT T Deus f E LH M fma M hema testes few mw mp s Investigator ProGest 3 28 01 79 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Customize a description of timer information in the text field Select the desired timer Enter a delay time in hours minutes and sec onds if desired Wait for a timer This step is a companion to the Start Timer step When the method reaches this step processing will stop until the specified timer has expired reached zero It is important to ensure that the timer number you specify in the Wait for Timer step matches that which you started in the Start Timer step Time Delay This step directs the system to wait at this point in the protocol A delay step will stop the processing of the method for the specified amount of time Nothing else will happen during the delay period The wait time can be entered in hours minutes and seconds 80 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest
26. the dilutor syringe on page 91 for more information about these selections The Autosampler Control allows the user access to check the nitrogen valve and heater control Refer to Check Nitrogen Valve on page 97 and Check Heater on page 98 for more information Other options under Autosampler control refer to robotic arm movements and should be accessed by service per sonnel only due to the possibility of damage to the probes Network Address allows the user to scroll through the network addresses to select the correct address for net working multiple instruments on the ProGest Refer to Network Address on page 99 for more information 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation Switching the system off The instrument stops operation after execution of the pro gram and returns to the stand by mode The program fin ished message will be displayed If you do not intend to use the instrument for some time you may want to switch it off entirely 1 Switch off the system with the On Off switch 2 Remove the reaction and collection racks dis pose of reagents 3 Empty waste bottle and dispose of solvents according to institution or national standards 4 Clean the instrument as described in the mainte nance section Emergency STOP In case of an emergency press the On Off switch located at
27. the contents of a single vial Refer to Pipette from one vial to another on page 77 for more information Investigator ProGest 3 28 01 65 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Rinse needles directs the system to rinse the needles both inside and out Refer to Start a timer directs the system to start a selected timer but allows the method to continue running Refer to Start a timer on page 79 for more information Time delay directs the system to wait at this point in the protocol Refer to Time Delay on page 80 for more information Wait for timer is a companion step to the Start Timer step When the method reaches this step pro cessing will stop until the specified timer has elapsed Refer to Wait for a timer on page 80 for more infor mation Wait for the operator this step directs the system to pause and wait for the operator Refer to Wait for the operator on page 81 for more information Common Parameters Several of the steps listed have similar parameters that need to be entered for the step to be used Some of these common parameters are discussed below rather than under each step to minimize repetition Diluter This screen is found under several steps such as Aliquot to each sample well Incubate purge aliquot delay Pipette from one vial to another and Ri
28. there is a seal made between the film and the red tray However do not over tighten as that may cause the reaction wells to fail to seal completely If loading the reaction wells manually 1 Remove the Reaction Plate Cover by removing the four corner screws holding the Reaction Plate Cover onto the Reaction Block 2 Load the red colored Reaction plate before plac ing in the Reaction Block in the following order Place a blank control at the beginning of the sample run i e a reaction well with gel matrix but no protein sample Blank controls are use ful on high sensitive runs to identify enzyme peptides derived from autodigestion Investigator ProGest 3 28 01 21 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation Place your sample gels into the next available spaces in the red colored plate making sure to note the identity of all samples and their posi tion on the reaction tray Continue until all sample gels have been placed in the red colored Reaction plate Place a blank control at the end of the series of samples 3 When all samples have been put in the red col ored Reaction plate place the Reaction plate into the Reaction Block place the plastic film onto the reaction plate and replace the cover 4 Secure the Reaction Plate Cover using the four mounting screws at the corners as stated previ ously Note Do not overtighten as t
29. 3 71 153 1419169 89175 FO ila 2045 03 639 95 1880 92 1907 93 76 1791 87 Pl 87 TIT ihr ih h AAA linus m 2211 1 2780 37 2487 67 d 33 faa Apt Mi vorn avian Search Results 1 12 28 matches 42 69293 9 Da pI 5 82 Acc P02769 BOVIN SERUM ALBUMIN PRECURSOR m z submitted 927 4973 1163 6161 1283 7136 1305 7061 1439 8190 1479 8015 1567 7508 1639 9482 1687 8698 1791 6697 2045 0344 2278 0305 B 124 MH Delta matched ppm 927 4940 3 6195 1163 6312 12 9893 1283 7112 1 8724 1305 7167 8 1149 1439 8123 4 6690 1479 7960 3 7041 1567 7433 4 7811 1639 9383 6 0576 1687 7862 49 5094 1791 7100 22 5196 2045 0285 2 8714 2278 2310 87 9861 Fig 5 start 161 66 361 402 360 421 347 437 118 267 168 1 end 167 75 371 412 371 433 359 451 130 280 183 19 Peptide Sequence K YLYEIAR R K LVNELTEFAK T R HPEYAVSVLLR L K HLVDEPQNLIK Q R JRHPEYAVSVLLR L K LGEYGFQNALIVR Y K DAFLGSFLYEYSR R R KVPQVSTPTLVEVSR S K QEPERNECFLSHK D K ECCHGDLLECADDR A R RHPYFYAPELLYYANK Y JMKWVTFISLLLLFSSAYSR G 1 Met ox Mass spectrum of the peptide digest from sample 5 obtained using the ProGest Validation kit 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com A rtisan Artisan Technology Group is you
30. 88 SOURCE www artisantg com Chapter 2 Operation e Acetonitrile ACN 100 used for washing and shrinking the gel pieces Note This solu tions should be stored in glass Ammonium Bicarbonate ABC 50mM used for buffering the reactions Ammonium Bicarbonate ABC 25mM used for buffering the reactions Reagents 10mM Dithiothreitol DTT in either 25mM or 50mM ABC used for reducing the disul fide bridges in protein e 100mM Iodoacetamide IAM in 50mM ABC used for alkylating the free Cys SH groups Enzyme e Trypsin 20ug used for enzymatically frag menting the proteins Loading the work area The ProGest has a number of containers for the liquids that are used during the digestion process Investigator ProGest 3 28 01 17 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation v a m 2 y s P pe cm i F a hy y Solvent Solvent Solvent Solvent Solvent j 18 uf i Enzyme A F7 Jd j B ANCA RD A RES X EZ AN an i ear bm E E i Solvent meg ee HOW qe or ol QUA dU LIE v PEL CN Enzyme Enzyme Enzyme RgtA RgtB B C D The vial locations are assigned specific letters which relate to their location on the ProGest reagent area For reagent placement used in the protocols provided with the ProGest refer to Appendix A Trypsi
31. Black no wells selected Gray some wells selected 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation 7 When you are finished select Done to return to the Main menu Priming Before starting the run it is necessary to prime the dilu tor This fills the syringe and tubing from the reservoir with fluid and purges air 1 Select Plate Configuration from the keypad 2 Select Setup from the keypad Investigator ProGest 3 28 01 29 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation EEE Press Prime Dilutor to start priming The probes will be placed over the rinse port and the syringes will fill and empty Check for bubbles in the syringes or tubing Con tinue priming until no bubbles are present Once pressed the Prime Dilutor key will change to Stop Dilutor Press Stop Dilutor to stop the priming The instrument will complete its prime sequence and return to the default position Select Done to exit the window and return to the Main menu Start Digestor 1 Press Start Digester to execute the digestion program A window will appear asking the user if they would like to delay the start of the run 30 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE w
32. F 1480 8615 1480 8640 1 7076 88 100 R IRFIAEGHPLSLK F 1538 6903 1538 7062 10 2992 131 143 KJ IGENKDAMDGWFR L 1554 6880 1554 7011 8 4403 131 143 K IGENKDAMDGWFR L 1Met ox 1762 9313 1762 9339 1 5252 72 87 R NELDKGIGTIISSPYR I Fig 1 Mass spectrum of the peptide digest from Sample 1 obtained using the ProGest Validation kit B 120 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 842 51 99 54 Y wo 1018 51 979 477 1334 73 50 06 2852 5 Mbps mm ALLIUM Nia Mhama A AEE AN 1581 81 1738 9 1709 9 1685 93 636 43 2198 21 2141101 209 E Appendix B ProGest Validation Kit 2584 13 2910 37 2807 33 3167 54 Search Results 1 8 24 matches 33 28982 7 Da pI 6 40 Acc P00921 BOVIN CARBONIC ANHYDRASE II EC 4 2 1 1 CARBONATE DEHYDRATASE II m z submitted 979 4771 1018 5098 1581 8137 1709 9011 2098 85 19 2198 2097 2852 5009 2868 4453 Investigator ProGest MH matched 979 4849 1018 4958 1581 8178 1709 9127 2098 8776 2198 2185 2852 4769 2868 4718 Delta ppm 7 9683 13 7518 2 5899 6 8131 12 2459 4 0186 8 4067 9 2435 Fig 2 36 56 226 250 226 250 Peptide Sequence K DGPLTGTYR L K DFPIANGER Q K YAAELHLVHWNTK Y K KY AAELHLVHWNTK Y R MVNNGHSFNVEYDDSQDK A K AVVQDPALKPLALVYGEATSR R R TLNFNAEGEPELLMLANWRPA QPL
33. Invert the vial sev eral times to mix well Keep at 4 C until use This solu tion is stable for 24 hours at 4 C Prepare Trypsin Enzyme C To prepare Trypsin Enzyme open the small vial G con taining Trypsin Resuspension Buffer Add 300 ul of Trypsin Resuspension Buffer to the 20ug lyophilized 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Artisan Technology Group Quality Instrumentation Appendix A Trypsin Digestion Kit trypsin powder Promega in the small glass vial pro vided in the zip lock plastic bag Draw the solution up and down in a pipette tip several times to mix well and then transfer the entire volume into Trypsin Enzyme vial C Keep at 4 C until use Prepare Formic Acid Solvent E No further preparation is needed Use solution as pro vided Prepare Water Solvent F Add 30 ml of 18 megohm cm water to the empty Solvent vial F Vial Placement Place the prepared reagent vials in the ProGest sample work area as shown in Fig 1 pu Tm a p a P SET AN fr aer id Pis P h r pr SS eres sini m Dacor EP Pere nin i M ou Aou ee Nee Sell 7 o i ho cR I A Wwe ke WY d ss Fig 1 Investigator ProGest 3 28 01 A 115 Guaranteed 888 88 SOURCE www artisantg com Appendix A Trypsin Digestion Kit Running the Protocol Follow the instruc
34. K N R TLNFNAEGEPELLMLANWRPA QPLK N 1Met ox Mass spectrum of the peptide digest from sample 2 obtained using the ProGest Validation kit 3 28 01 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com B 121 Appendix B ProGest Validation Kit 1345 76 842 511 834 467 756 3 j 1242 i rem T aie 1597 73 1571 74 1425 76 1522 82 1687 87 182 Wi TEM 1859 2281 22 2008 98 1874 98 8 3337 8 3237 63 mv eom m REN 2326 34 2196 2476 34 2807 35 Search Results 1 8 24 matches 3396 42750 3 Da pI 5 19 Acc P01012 CHICK OVALBUMIN PLAKALBUMIN ALLERGEN GAL D 2 GAL D ID MH matched m z submitted 1345 7625 1345 7381 1571 7220 1571 7164 1597 7271 1597 7168 1687 8673 1687 8404 1859 0006 1858 9663 2008 9777 2008 9464 2281 2197 2281 1828 2476 3378 2476 3121 B 122 Delta ppm 18 1611 3 5389 6 4393 15 9714 18 4448 15 5981 16 1686 10 3678 start 370 187 264 127 143 340 end 381 199 276 142 158 359 85 104 159 Fig 3 Mass spectrum of the peptide digest from sample 3 obtained using the ProGest Validation kit 181 3 28 01 Peptide Sequence K HIATNAVLFFGR C K AFKDEDTQAMPFR V 1Met ox K LTEWTSSNVMEER K 1Met ox R GGLEPINFQTAADQAR E R ELINSWVESQTNGIIR N R EVVGSAEAGVDAASVSEEFR A R DILNQITKPNDVYSFSLASR L R NVLQPSSVDSQTAMVLVNAIVFK G 1 Met ox Investigator
35. OURCE www artisantg com Chapter 1 Introduction System Software The operating software for the Genomic Solutions ProGest is stored permanently on a single chip FlashDisk on the PC board within the controller Upgrades can be loaded manually by transferring files from a 3 5 1 44MB floppy disk or by means of the Method Editor See Upgrading Software on page 96 for information about upgrading software at the ProGest or Loading Software on page 54 for information about downloading software from the Method Editor to the ProGest Future ProGest software upgrades will be avail able from the Genomic Solutions website Investigator ProGest 3 28 01 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 1 Introduction System Hardware 1 Transfer Tubing 8 Waste Bottles 2 Dual Probes 9 Protective Cover 3 Dilutor Unit for Syringes 10 Reaction Block 4 Pipetting Robot Arm 11 Reagent Rack 5 Work Area 12 Collection Rack 6 Keypad 7 Dilutor Reservoir 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 1 Introduction 1 Keypad 4 Screen Adjustment Knob 2 On Off Switch 5 Connector for keyboard 3 3 5 Disk Drive System Overview The ProGest can be controlled from the touchpad on the ProGest itself Characters are digitally displayed on a Liquid Crystal Disp
36. aff and should be treated with care Protective gloves should be worn at all times to protect the user and to minimize possible ker atin contamination Reagent Preparation Note Use only high quality 18 megohm cm water for preparation of all solutions Use solutions immediately after preparation Investigator ProGest 3 28 01 A 113 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Appendix A Trypsin Digestion Kit A 114 Prepare Solvents C and D To prepare Solvent C 50mM Ammonium Bicarbonate resuspend the contents of Solvent vial C in 30 ml of 18 megohm cm water Invert the vial several times to mix well Keep at 4 C until use This solution is stable for 5 days at 4 C To prepare Solvent D 25mM Ammonium Bicarbonate resuspend the contents of Solvent vial D in 30 ml of 18 megohm cm water Invert the vial several times to mix well Keep at 4 C until use This solution is stable for 5 days at 4 C Prepare DTT Reagent A To prepare Reagent A 10mm dithiothreitol solution add 10 ml of prepared Solvent D 25mM Ammonium Bicarbonate to the contents of vial A Invert the vial sev eral times to mix well Keep at 4 C until use This solu tions is stable for 24 hours at 4 C Prepare lodoacetamide Reagent B To prepare Reagent B 100mM iodoacetamide solution add 10 ml of prepared Solvent C 50mM Ammonium Bicarbonate to the contents of vial B
37. ch instrument networked 82 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization ProGest Instrument In the screen above one ProGest instrument is networked to the PC To see more detailed information double click on the device in the network view The detail page will include information about the progress of a run if the instrument is running and steps to start the instrument from the PC if it is not running Investigator ProGest 3 28 01 83 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization pensis Dema orate COIT Deralma DEMTD Desde Butea Debe METRE rera mesie laura 7 Phn epu Fije ii biai keine Flach a uas pasam Marcus ran Er Controlling ProGest Remotely From the Method Editor it is possible to start stop pause the system and select sample positions 84 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Desk Dema indomita moi Lo Bare Demxmem ff reunion iar 7 Leni ahai Tan Fog Start ProGest Remotely To start the ProGest from the Method Editor Click the right mouse button on the device in
38. chnology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols Step Number Description 11 Start Timer Starting Timer 1 Time 15 minutes 0 seconds 12 Neutralizing with ABC Source 50mM Bicarbonate Vol 50yl 13 Reaction Time Waiting for Timer 1 14 Shrinking gel Source Acetonitrile Vol 100u1 Hold Time 10 minutes 0 seconds 15 Set Heaters to High Heater HIGH 16 Reduction with DTT Source DTT Vol 30u1 Hold Time 10 minutes 0 seconds 17 Turn off Heaters Heater OFF 18 Reduction cooling Delay 20 minutes 0 seconds 19 Alkylation Source Iodoacetamide Vol 30u1 Hold Time 15 minutes 0 seconds 20 Washing with ABC Source 50mM Bicarbonate Vol 40ul Hold Time 10 minutes 0 seconds 21 Washing with CH3CN Source Acetonitrile Vol 50u1 Hold Time 15 minutes 0 seconds 48 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols Step Number Description 22 Shrinking with CH43CN Source Acetonitrile Vol 50u1 Hold Time 15 minutes 0 seconds 23 Purge wells Source Acetonitrile Vol Oul Hold Time 10 minutes 0 seconds 24 Prepare enzyme Source 25mM Bicarbonate Vol 950u1 Destination Trypsin Vol 900u1 25 Mixing enzyme Source Trypsin Vol 600u1 Destination Trypsin Vo
39. describes the use of the Genomic Solutions ProGest during operation You will learn how to Turn the system on and off Prepare reagents Prepare the system for operation Load the work area e Start a run Stop interrupt or abort a run Preparing the system Empty waste bottle Note The chemicals in the waste bottles are hazardous material which must be disposed of properly Ask your safety officer and follow the regulations of your institu tion 1 Empty waste bottles 1 into appropriate reser voir or container Investigator ProGest 3 28 01 15 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation 16 2 Fill up the dilutor reservoir 2 with 18 MOhm water which has been filtered through a 0 45um filter and degassed Preparing reagents Before each run you will need to prepare solvents as fresh solutions It is recommended that the ProGest be used with ProGest Trypsin Digestion kit Cat No 70 0210 Refer to Appendix A Trypsin Diges tion Kit on page 113 for reagent preparation instruc tions For safety reasons be sure to wear safety glasses gloves and a protective suit or coat when handling sol vents The reagents and solvents listed below are used in the protocols provided with the ProGest Solvents 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888
40. elect an airgap volume in ul if desired Select an equalization time in seconds e Select a discard volume in ul Step Sequence specified air gap is aspirated needles move into source vial calculated volume of liquid is aspirated needles visit first pair of samples moving to a lower purge position the nitrogen valve is opened for a specified time then needles rise to their normal dispense position specified volume of liquid is dispensed com plete with droplet aspiration if specified Note Refer to page 68 for more information sequence repeats for all active samples specified discard volume is dispensed into the drain a rinse is performed similar to rinse step processing halts until specified hold time has elapsed Note The specified hold time starts at the beginning of the step rather than the end it includes the time necessary to perform all the purging and dispensing and the final rinse Investigator ProGest 3 28 01 75 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Move the sample rack This step directs the system to move the sample rack to a designated location The ProGest operates with the rack in two positions The first to the left of the instrument is used when liquid purged from the sample wells is to be discarded into the drain The second position to the r
41. function This chapter will give you an overview of some possibly occurring failures and solutions of the system Machine Test Method A quick check on the correct operation of the ProGest would be the Machine Test Method This is found under the Method list on the instrument This method will dispense solvent 50u1 into the 96 well reaction plate in the reaction block A further quantity of another solvent is then added to the reaction plate The system stops and asks the user to check for any drips by looking under the bottom of the tray don t move it before pro ceeding The plate will be moved to the right hand side of the work area It is then purged with nitrogen and the liq uid collected in the collection plate underneath There should be equivalent volumes in every well To set up the system for the Machine Test 1 Add distilled water to Solvent positions A and D Investigator ProGest 3 28 01 101 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 6 Troubleshooting 2 Be sure to place both a sample plate with pierced sealing film and a collection plate on the instru ment Power the system on Select Machine Test Method from the protocol list for the desired method Select Plate Configuration to select the desired sample positions Refer to Plate Configuration on page 27 for more information It is usual to select all for this test
42. g set tings from the previous run Method Plate Configuration Start Delay To perform a run according to the previous settings no changes to the settings need to be made For a first time Investigator ProGest 3 28 01 25 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation user or to change the existing settings follow the instruc tions in Method Selection Method Selection The first step is to load a digestion method if none has been previously selected 1 Press Method on the keypad A list of available methods will appear 2 Select the desired digestion method Note The protocols provided are listed step by step in Chapter 3 Protocols and how to edit or create protocols is covered in Chapter 4 Method Edi tor Note Machine Test is a maintenance func tion and is not a digestion method 26 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation Plate Configuration Once the digestion method has been selected the next step is to set up the plate configuration which wells of the plate are to be processed Select Plate Configura tion from the keypad 1 A screen will appear with a schematic of a Reac tion plate divided into four parts Quadrants selected for processing are shown white unse lected quadrants in black
43. g the eluted peptides 33 Set UD rr 33 Switching the system off 04 35 Emerg n cy STOP 2 6e20565554 eeaeewsecrxsiee 6 35 3 Protocols wtctudad a PE E Ra RE RUE Rod aw 37 4 Method Editor 53 Method Editor Overview 0 00000 eae 53 Loading Software 2o da he RR ARAS eae 54 Method Editor Window eleeeeeese 54 Menu Items and Tool Bar 02004 56 PUG FC MD 56 New aisessqeuibodeahecisess Be sdedcsd erra 56 S MERCI MEE Ur EP 58 DAVE uwibxdqamedqeacaquaxadu rss qq Esa Ps d 58 SUVS AS ore aT ROC LO d ERI ge degunt do dd aC Y e 59 Set Method Title uesssasddkerkeisskerx nes 59 Pant SxeGke doce uSGeseero Seed see she RENES 60 ii 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Contents Print PIevieW oues eu oko veREXR EAR IX RE PER 60 Print SOUP 2456 debo a eae debo s eroe deba 60 lo cd Pha ehe ee arant 61 Delete Sep descsxs cixssmpX x GC Genes R3 61 Insert Step ga addc ad pore pale acr s Rad 62 Append Step lt cadeeersdrsdeuatees Secures Add 63 Edit Vial Contents Names 04 63 List Of Steps pocece c EGXaVRREReTEe e Yd EUR ERES Kus 65 Common Parameters 244 1422 cos RR RR Res 66 Dil ter c 66 RUSS kdisseexexkises8RXeEWdd eEe Ri seus 68 SIOUIGE Luosesoi da oie do he EA Id x ie os ceed 69 Aliquot to each samp
44. hat may cause the reaction wells to fail to seal completely Preparing the Reaction Plate Once the reaction plate with the plastic film cover has been placed on the reaction block and secured using the four corner screws holes need to be pierced into each of the 96 wells prior to running the instrument A small metal rod is included with the system Using the small tool gently pierce the plastic covering each of the wells as shown pushing the tool down as far as it will go Itis recommended that you keep the tool clean and do not handle the tip Store it in a clean tube to keep the tip sharp This is important as it ensures the hole gives a good seal to the probes 22 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation Removing the Reaction Block For easier operation you can remove the entire Reaction Block from the sliding frame but it is not necessary 1 Move the Reaction Block to the left end of the sliding frame 2 Liftthe left side until the alignment pins are visi ble 3 Move the block over the rightmost position and lift the right side Be sure not to damage the four alignment pins at the corners 4 Replace the Reaction Block in the work area by reversing this procedure Make sure the block slides freely on the sliding frame The Reaction Block should be positioned centrally on the slid Investigat
45. he reservoir equalization time and dis card volume should also be entered Aqua Phe Goo ee E M ae A C f 5 aa oe lum P Da 3 70 Aliquot Customize the step with a description to show what is being performed This description is displayed on the ProGest screen when this step is being executed Enter the reagent source of the aliquot to be added to each sample well from the drop down menu list Reservoir allows liquid from the res 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization ervoir to be added This selection changes a valve in the dilutor to allow liquid to be aspi rated directly from the reservoir bottle the sample probes do not move to the reservoir bottle to aspirate the liquid e Enter the desired amount of reagent in ul to be added Dilutor Select the desired aspiration and dispense time in ml min default speed 10ml min e Select an airgap volume in pl if desired The air gap is adjustable 10 ul is a typical value Select an equalization time in seconds e Select a discard volume in ul Step Sequence specified air gap is aspirated needle moves into source vial calculated volume of liquid is aspirated needles visit pairs of sample wells and dis pense specified amount of liquid specified discard v
46. hnology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols Step i dy Nubes Description 27 Mixing enzyme Source Trypsin Vol 600ul Destination Trypsin Vol 600u1 28 Adding Enzyme to samples Source Trypsin Vol 15ul Hold time 10 minutes 0 seconds 29 Moving rack for extraction Start X 400 Y 925 Z 80 Stop X 1642 Y 925 30 Moving rack for extraction Start X 1610 Y 925 Z 80 Stop X 1642 Y 925 31 Set Heaters to 37 C Heater LOW 32 Digestion first stage Delay 1 hour 30 minutes 0 seconds 33 Rinse needles Inside 1000u1 Outside 1000u1 34 Adding water to gel Source Water Vol 10ul 35 Digestion second stage Delay 2 hour 0 minutes 0 seconds 36 Rinse needles Inside 2000u1 Outside 2000u1 37 Adding ABC Source 25mM Bicarbonate Vol 10ul 38 Extracting peptides Delay 10 minutes 0 seconds 40 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols Step N mbr Description 39 Adding CH4CN for extraction Source Acetonitrile Vol 20u1 40 Incubation with CH3CN Delay 10 minutes 0 seconds 41 Extracting with Formic Acid Source 10 Formic Acid Vol 20u1 Hold Time 10 minutes 0 seconds 42 Start Timer Starting Timer 1 Time 15 minutes 0 seconds 43 Adding CH4CN for ext
47. hod step x Chone the hype ol step you wart bo add aliquot io eadh gasps mei Arpani to each campos by 13 Incubale page alquor dela Howe the sample rack Fipeite nom one vial bo smother Rinse needles Shack a bri Tira chilin 2 Select highlight the desired step to insert from the drop down menu list Select OK 3 The step will be inserted at the selected step num ber in the protocol and the instructions originally at that number will be moved to the next step number For example if step 5 now is the step Heater con trol and is selected when the step Rinse needles is inserted it will become step 5 and Heater control moves to step 6 62 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Append Step This command allows the user to add or append steps to an opened protocol 1 Select Append Step from the Edit menu The New Method Step window appears New method step x Choose the type of step you want to add Incubate purge aliquot delay En Tu Move the sample rack J Pipette from one vial to another Rinse needles Start a timer Time delay Wait for a timer Wait for the operator 2 Select highlight the desired step to insert from the drop down menu list Select OK 3 The selected step will appear as the
48. ight is used when it is important to catch the liquid purged from the wells into another plate Caution It is strongly recommended that you use the standard moves as it is very easy to perform rack move operations that may cause damage to the ProGest Lam T em mt cua kar en ie Fui ad Tobces iate Lcd BIS ie LN en Tin rl T Caen tem oi Dai nitri i ftn T Lime cmd i Gin ew Select the type of move desired from right side left side from left side to extraction position or custom move The recommended sequence is to move the rack to the left position then to move the rack to the extraction position Collect sample rack from right hand side moves the rack from any position at the right of the instrument into its starting position This should normally be followed by a Collect from left step Collect sample rack from left hand side moves the rack from any position at the left of 76 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization the instrument to the correct starting position Move to extraction position this step assumes the rack is in the correct starting posi tion and then moves it to the right hand extrac tion position Note After the move the system needs to know that the samples are now in a different position for this reason the update sample positio
49. l 600u1 26 Mixing enzyme Source Trypsin Vol 600u1 Destination Trypsin Vol 600u1 27 Mixing enzyme Source Trypsin Vol 600u1 Destination Trypsin Vol 600u1 28 Adding Enzyme to samples Source Trypsin Vol 15ul Hold time 10 minutes 0 seconds 29 Moving rack for extraction Start X 400 Y 925 Z 80 Stop X 1642 Y 925 30 Moving rack for extraction Start X 1610 Y 925 Z 80 Stop X 1642 Y 925 Investigator ProGest 3 28 01 49 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols Step e Number Description 3l Set Heaters to 37 C Heater LOW 32 Digestion first stage Delay 1 hour 30 minutes 0 seconds 33 Rinse needles Inside 1000u1 Outside 1000u1 34 Adding water to gel Source Water Vol 10pl 35 Digestion second stage Delay 6 hour 0 minutes 0 seconds 36 Rinse needles Inside 2000u1 Outside 2000u1 37 Adding ABC Source 25mM Bicarbonate Vol 10ul 38 Extracting peptides Delay 10 minutes 0 seconds 39 Adding CH3CN for extraction Source Acetonitrile Vol 20u1 40 Incubation with CH3CN Delay 10 minutes 0 seconds 41 Extracting with Formic Acid Source 10 Formic Acid Vol 20u1 Hold Time 10 minutes 0 seconds 42 Start Timer Starting Timer 1 Time 15 minutes 0 seconds 43 Adding CH3CN for extraction Source Acetonitrile Vol 20u1 50 3 28 01 Inve
50. lay from a computer by means of a serial port connection from the RS485 interface on the ProGest using the Method Editor software program The touchpad contains display and function information It allows the user to select from methods stored on the ProGest itself or on a 3 5 floppy disk select the desired sample locations on the 96 well plate and start delay a start run pause or abort the method Investigator ProGest 3 28 01 5 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 1 Introduction The ProGest can also be run by means of networking through a cable connection from the ProGest to your PC PC Specifications for ProGest Method Editor software Windows 95 98 or Windows NT 4 0 or later RS232 port for cable connection to ProGest 2 MB disk space Color Monitor minimum screen resolution 600 x 800 Using the Method Editor software program on your PC allows for convenient method transfer instead of storing method files on a floppy disk and transferring them manually to the ProGest they can be sent to the ProGest by means of the Method Editor and stored on your PC s hard drive Refer to Method Transfer on page 86 for more information remote supervision the Method Editor software can supervise a number of Genomic Solutions instruments such as ProGests and ProMS s simultaneously The status of each is shown in the network view of
51. le Cause Solution Green LED control light at the right arm side of the robot does not light up 1 Power cord unplugged 2 Blown fuse 1 Check the power source 2 Call Technical Service Green LED of dilu tor does not light up 1 Power cord unplugged 2 Blown fuse 1 Check the power source 2 Call Technical Service Display of keypad stays dark 1 Contrast button on front of instru ment turned 2 Power cord unplugged 3 Blown fuse 1 Adjust contrast with button on front of instrument 2 Check the power source 3 Call Technical Service Dilutor syringe leaks A small leak can develop after sev eral months of operation 1 Dismantle the syringe and clean 2 Change the syringe 104 3 28 01 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Investigator ProGest Chapter 6 Troubleshooting Table 1 Problem Possible Cause Solution Dilutor valve leaks The dilutor valve has a safety outlet at its left side If the probe is blocked the solvent can leak through this outlet Remove and check the probe See Probe Adjust ment on page 94 2 Check the trans fer tube for bends and clogging Robot stopped dur Power failure Check the power ing execution source Investigator ProGest 3 28 01 105 Artisan Technology Group Quality Ins
52. le well 70 Heater Control 2 duse yp as RI e a 72 Incubate purge aliquot delay 12 Move the sample rack ssssus 76 Pipette from one vial to another 77 Rinse needles 2 2 09x ernir r EE REPE PE 79 Start 8 timer iussa hes eus E race ah e d pd 79 Wait fora timer 42 2 Le ERR RR ER bends s 80 Time Delay 5a useo dd o Xy ce REPE DEREN SONS 80 Wait for the operator 0 0 00 e ee eee 81 ral P 82 SENES lt pradie CER Ex en da C ERR Sb ied 82 Network Status 2x 2er Re RR RI RARE RE ERTREES 82 Controlling ProGest Remotely 84 Start ProGest Remotely 000 85 Stop or Pause ProGest Remotely 86 Select Sample Positions Remotely 86 Method Transfer iudcesmecesexa dene ERO sind 86 Investigator ProGest 3 28 01 iii Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Contents 5 Maintenance issues nad nay Rn te REX we RR 89 General Safety Procedures 0 00055 89 Installation sogen eei oS rd nS each Y ure Gedy 91 Changing the dilutor syringe lesse 91 Probe Adjustment lt s 220 2snddanesadeeasd tanec 94 Priming the dilutor 52 2 ee cease inked esate eek 96 Upgrading Software 0 0 0 eee eee eee 96 Autosampler Control 42 232 200a0 0240424954004404 97 Check Nitrogen Valve
53. ly saved 1 Select Save As from the File menu The Save As window appears in which you can name a directory and file in which to save the protocol as a digestion method met Set Method Title This command allows the user to set or give a descriptive title to a method 1 Select Set Method Title from the File menu The Method Title window appears Enter a description of the method or aris Core 2 Type in the desired description title of the method and select OK when finished The title will appear in the title bar of the opened window of the Method Editor in parenthesis next to the file name and Investigator ProGest 3 28 01 59 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization 60 will be printed along with the file name if a proto col is printed Note The Method Title is what will appear on the ProGest method list It is rec ommended that you give each method a differ ent title so that you will be able to distinguish them once they are loaded onto the ProGest Print This command allows the user to print a copy of the opened protocol 1 Choose Print from the File menu The Print win dow appears in which you can select your printer information 2 Select OK to print the protocol Print Preview This function allows the user to view the layout of the printed image prior to actually printing it
54. minutes 0 seconds 43 Extracting with Formic Acid Source 10 Formic Acid Vol 20u1 Hold Time 10 minutes 0 seconds 44 Start Timer Starting Timer 1 Time 15 minutes 0 seconds 45 Adding CH3CN for extraction Source Acetonitrile Vol 20u1 46 Extraction with CH3CN Waiting for timer 1 47 Shrinking with CH4CN Source Acetonitrile Vol 30u1 Hold Time 15 minutes 0 seconds 48 Purge wells Source Acetonitrile Vol Oul Hold Time 10 minutes 0 seconds 49 Rinse Inside 2000u1 Outside 2000ul 50 Turn off heaters Heater OFF 46 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols Long Trypsin Digestion Default2 met Step Number Description Moving rack to run position Start X 2100 Y 925 Z 110 Stop X 447 Y 925 Moving rack to run position Star X0 Y 925 Z 110 Stop X 93 Y 925 Moving rack to run position Start X 300 Y 925 Z 80 Stop X 419 Y 925 Pierce Sheet Source Reservoir Vol Oul Hold Time 0 seconds Turn off Heaters Heater OFF Initial Rinse Inside 2000u1 Outside 3000p1 Washing with Bicarbonate Source 25mM Bicarbonate Vol 100u1 Hold Time 0 seconds Soak in ABC Delay 20 minutes 0 seconds Purging ABC Source Acetonitrile Vol Oul Hold Time 0 seconds 10 Washing with CH3CN Source Acetonitrile Vol 50ul Investigator ProGest 3 28 01 47 Artisan Te
55. n Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Equalization time is selected in this screen Because the air gap is elastic it may be necessary to allow some set tling time after the dilutor has operated This parameter is know as equalization time and is adjustable for each step A typical value is 1 second Discard volume is also selected in this screen An addi tional safeguard against contamination is to draw up more fluid than is necessary dispense the specified amount and then discard the remainder Steps such as aliquot and incubate have a parameter which speci fies a discard volume When using a pipette step this effect may be achieved by setting the aspiration volume higher than the dispense volume Droplet aspiration is another option that may or may not be present under Dilutor depending upon the step chosen When present this feature is used to improve fluid delivery accuracy When dispensing to the sam ple for example slightly more liquid is dispensed than specified by the dispense volume alone The syringe then draws back by this amount to collect the droplet at the tip of the needle This parameter is normally set to Zero Rinse This screen is found under two steps Incubate purge aliquot delay and Rinse needles 68 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88
56. n must be set If you do not select it on this move the ProGest will move the sample rack to the right but continue to visit the positions on the left to dispense to the plate Caution Do not perform this move unless you know that the rack is already in the normal starting position Enter custom move settings in mm 10 that is tenths of a millimeter for start position and end position this step allows for complete flexibility over the move Caution must be used when experimenting with custom moves Update sample position after move this option is normally used when performing the move to extraction position It tells the ProGest that after the move has been executed it should expect to find the sample plate in a new posi tion The ProGest always starts by assuming that the samples are in the left hand position Pipette from one vial to another This step directs the system to transfer liquid from one vial to another It can also be used to stir the contents of a single vial Investigator ProGest 3 28 01 71 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Pete Dind x n mara Ere eae er aei ia ie a M pw inn uU linum i ni Lig rinm 5 3 wx l a ee ne mam Tm m Gpp a Pipette e Customize the step to show which reagent is being added to which destination in the description text field
57. n Digestion Kit on page 113 Solvent A This position is used for the Acetonitrile as it will hold a 100ml glass bottle Solvents B through F These positions are used for other solvents such as They will hold 30ml plastic vials Note Be sure to check which positions are selected for your protocol by checking Vial Contents Names under the Edit menu in the Method Editor software Reagent A DTT This will hold a 10 or 15ml plastic vial Reagent B IAM This will hold a 10 or 15ml plastic vial Enzyme A These positions can be used for enzyme solu tions They will each hold a 0 5ml microfuge tube 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation Enzyme B These positions can be used for enzyme solu tions They will each hold a 2ml microfuge tube Enzyme C This position can be used for enzyme solu tions This will hold a 10ml plastic tube Enzyme D This position can be used for enzyme solu tions This will hold a 10ml plastic tube If you are using the one of supplied protocols be sure to check the proper placement of the reagents using the Method Editor See Appendix A Trypsin Digestion Kit on page 113 Isolating protein samples The proteins to be analyzed are usually separated by 1D or 2D polyacrylamide gel electrophoresis PAGE After staining the protein features can be excised from the gel
58. n and follow the instructions in the ProMS operator s manual to spot the MALDI plate If spotting onto a MALDI plate manually add 1 2 ml of the supernatant from the matrix mix ture directly to the dried peptides and spot onto the MALDI plate MALDI TOF Results The results that can be expected from mass spectrometry of the standard peptides on a MALDI TOF mass spec trometer are shown in Figures through 5 The illus trated spectra were acquired on a PE BioSystems Voyager DE RP PRO MALDI TOF mass spectrometer in reflectron mode The approximate mass accuracy was Investigator ProGest 3 28 01 B 119 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Appendix B ProGest Validation Kit 20ppm The database searches were conducted using Pro tein Prospector software and using the Swiss Prot sequence database 1762 93 1163 61 1554 69 1538 69 1211 65 1480 86 1432 8 2211 1 2032 1 2807 33 828 445 NU 277 67 s i 4 612 7 3215 34 TT Mi uud twi Mi linm wh id TT TH RPM TN Search Results 1 7 15 matches 4646 24005 4 Da pI 4 99 Acc PO1070 SOYBN TRYPSIN INHIBITORS A AND C PRECURSOR KUNITZ m z MH Delta submitted matched ppm start end Peptide Sequence 1163 6154 1163 6424 23 2444 77 87 K GIGTIISSPYR I 1211 6512 1211 6788 22 7978 90 100 R FIAEGHPLSLK F 1432 8139 1432 8276 9 5568 77 89 K GIGTIISSPYRIR
59. n up into 100p1 of 10 formic acid and automatically con centrated and desalted using micro reverse phase sample preparation techniques and spotted onto the MALDI tar get in matrix If spotting by hand the peptides can be taken up into a very small volume 10p1 of formic acid then concentrated and desalted using micro reverse phase sample preparation techniques and spotted onto the MALDI target in matrix General Safety Procedures The instrument must be operated by trained personnel only It is important to know and understand safety reg ulations regarding the instrument and the contents of the operator s manual Chemicals must be handled with care Please observe the current national safety regulations regarding the use and disposal of the following chemicals Acetonitrile ACN Ammonium Bicarbonate ABC Formic Acid HCOOH Dithiothreitol DTT e Todoacetamide IAM 10 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 1 Introduction Enzymes Wear safety glasses gloves and a lab coat during reagent preparation and instrument set up If accidental spillage occurs carefully clean up the spillage taking into account the nature of the spilled liquid and the nec essary precautions Prepare reagents in a fume hood During operation the instrument generates vapors which pose a health hazard The ProGest
60. ns STOP the system should be powered off by the On Off switch and the power cord disconnected from the power outlet General Safety Procedures The instrument must be operated by trained personnel only It is important to know and understand safety reg ulations regarding the instrument and the contents of the operator s manual Chemicals must be handled with care Please observe the current national safety regulations regarding the use and disposal of the following chemicals Acetonitrile ACN Ammonium Bicarbonate ABC Investigator ProGest 3 28 01 89 Guaranteed 888 88 SOURCE www artisantg com Chapter 5 Maintenance 6 90 Formic Acid HCOOH Dithiothreitol DTT e odoacetamide LAM Enzymes Wear safety glasses gloves and a lab coat during reagent preparation and instrument set up If incidental spillage occurs carefully clean up the spillage taking into account the nature of the spilled liquid and the nec essary precautions Prepare reagents in a fume hood During operation the instrument generates vapors which pose a health hazard The ProGest should only be operated with the cover closed in a well ventilated location Please consult your organization s safety reg ulations as to whether this instrument should be ope arted in a specially designated area or fume hood e Warning Please make sure that nothing interferes with the movement of the instrument parts Nothing
61. nse needles 66 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Apes Dum Ape goes oed mW siae Dimmi o ntti Aap rires Po Emsan fia Chemie D 0n n this screen the user can select aspiration and dis pense speed of the selected liquid This value is in ml min Choosing an appropriate speed for the dilutor is important When rinsing a high speed is necessary to achieve a good washing action When handling fluids with an air gap care must be taken to preserve the air gap The speed at which the air gap is damaged depends upon the volume of the air gap the diameter of the transfer tube between the needles and the syringe and the properties of the liquid being transferred Typi cal working values for aspirate and dispense speeds are 5 10 ml min Air gap volume is also selected in this screen This value is in ul Because the same needles and pumps are used for all the fluid handling within the system care must be taken to avoid contamination Before drawing any liquid into the needles aspirating an air bubble should be created in the tube This separates the reser voir liquid always present in the tube from the liquid being transferred The air gap is adjustable 10ul is a typical value Investigator ProGest 3 28 01 67 Artisan Technology Group Quality Instrumentatio
62. oGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation Handling the eluted peptides Once the digestion program is finished the eluted pro teins may be removed for further analysis 1 Slide the Reaction Block to the left 2 Remove the blue Collection plate containing the eluted peptides and label it as needed for future identification 3 To prepare the peptides for further analysis dry the solution by freeze drying or using a vacuum centrifuge Set Up This window allows the user access to some of the set up and troubleshooting functions available in the ProGest such as priming loading a syringe upgrading software selecting addresses for networking nitrogen and heater check Except for priming these functions would not be used routinely during operation only during network setup maintenance or troubleshooting To access this screen 1 Power up ProGest instrument if not already run ning 2 Select Plate Configuration from the keypad 3 Select Setup from the keypad The Engineering Options window will appear Investigator ProGest 3 28 01 33 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation 34 The Engineering Options are covered in Chapter 5 Maintenance Refer to Priming the dilutor on page 96 Upgrading Software on page 96 and Changing
63. ock to the connection under the workstage 2 Check the reac tion block Check Heater on page 98 If not working contact Technical Services No peptides found 1 Too little protein 1 Run control loaded digests of a known protein such as BSA at different concentrations using freshly pre pared reagents 2 Wrong reagents 2 Check reagents and buffers 3 Enzyme not 3 Check enzyme working 4 Wrong method 4 Check method selected Note Machine test does not do digestion 5 Problems in 5 Check probes reagent delivery 110 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 6 Troubleshooting Table 1 Problem Possible Cause Solution Too many wrong peptides found 1 Contamination This is most likely if the contaminat ing peptides can be assigned to a pro tein such as kera tine 2 More than one protein in the gel piece isolated 3 Incomplete digest of a very large protein 4 Heavily modified protein which is difficult to identify Repeat the experi ment with care fully cleaned material and include control pro teins for compari son Technical Assistance If you need technical assistance and you are in North America or Europe contact the Ann Arbor Michigan office Telephone Toll Free 1 877 GENOMIC 436 6642
64. of the instrument Note Be sure to pierce the film sheet Press Start to start the system Do not select a Delay Start Time The system will home itself and then start the process The following steps should occur in the following order If any step does not occur as described or if the system halts during the run call Technical Service for assistance Steps 102 3 28 01 Rack containing 96 well reaction plate moves to the run position left hand side Film is pierced The heater turns off Rinse needles inside and out System dispenses 50u1 of Solvent A into the reaction plate The heater switches to High System dispenses 50u1 of Solvent D into the reaction plate The method pauses now to allow the user to examine the dispensed liquid in the sample Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 6 Troubleshooting tray if required Press OK to continue the method Sample plate moves to the right hand collec tion position for extraction Reaction plate is purged with nitrogen The heater is turned off Wait while run finishes Check for liquid in the 96 well collection plate Investigator ProGest 3 28 01 103 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 6 Troubleshooting Troubleshooting Tips Table 1 Problem Possib
65. olume is dispensed to the drain Note When the instrument is processing many wells it aspirates the maximum volume dispenses to the first wells refills and continues dispensing to subsequent wells Investigator ProGest 3 28 01 71 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Heater Control The ProGest Reaction Block is fitted with a heater con trolled by a temperature controller built into the front panel The heater can be set to two different temperatures within the method The Heater Control step directs the system to turn heater to low 37 C or high 60 C or turn the heater off ambient Incubate purge aliquot delay This step directs the system to purge one liquid from a sample tube and replace it with another The delay or hold time is provided to allow time for a reaction to take place before the instrument moves on to the next step 72 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Artisan Technology Group Chapter 4 ProGest Method Editor Customization ncubate Dilutor Investigator ProGest Customize the step with a description to show what is being performed This description is displayed on the ProGest screen when this step is being executed Enter the reagent source of the aliquot
66. onto it into the disk drive on the front of the instrument Note Upgrades to the software will be accessible to be down loaded from the Genomic Solutions website Copy the downloaded file to a blank formatted 1 44MB 3 5 floppy disk 1 Select Plate Configuration from the keypad 2 Select Setup from the keypad 3 Insert the floppy disk with the new software upgrade on it into the disk drive at the front of the ProGest instrument 96 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 5 Maintenance 4 Press Upgrade SW 5 Follow the instructions on the display Press OK 6 To access the new software power the ProGest off at the main power switch front of instru ment remove the floppy disk from the disk drive and then turn the ProGest back on again Autosampler Control Some of the functions of the Autosampler are found under the Setup menu under Plate Configuration These allow the user access to a nitrogen valve check and a heater check Note Autosampler functions that pertain to the robotic arm Ref 1 Go XY Go Z and Free should be accessed by service personnel only If you encounter any problems with the movements of the robotic arm please contact Genomic Solutions Technical Service for assis tance Check Nitrogen Valve After a long period of inactivity or when there is a sus pected malfunction it is possible
67. or ProGest 3 28 01 23 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation ing frame It is moved to the correct position by the needle at the beginning of the run Load the collection rack Collection Plate ee ue erer EFC w we us Ue ue e han ee ee AL LIAJG LZ 11 1 L1 Once the samples have been placed in the Reaction Block and covered a blue colored Collection plate must be placed onto the collection block Switching the system on During operation of the ProGest organic vapors are gen erated For safety reasons the following conditions must be observed during the system operation operate only with protective cover closed make sure nothing hinders the movement of the instrument arm and probes 1 Verify that the power cord is plugged in 24 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation 2 Close the protective cover making sure not to catch the cable connecting the heater block 3 Adjust the nitrogen pressure to 15psi 1bar Once 15psi is acheived allow 15 minutes for the regulator valve to stabilize 4 Switch the system ON with the On Off switch at the front of the instrument The system will boot up and then the main menu will appear Settings The instrument automatically saves the followin
68. peptide fragments generated by trypsin digestion the most com monly used enzyme can be considered to be a finger print for each protein The masses of each peptide fragment from a protein fingerprint can be accurately Investigator ProGest 3 28 01 7 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 1 Introduction determined by mass spectrometry and these fragment sizes can be used to search against a variety of databases for a match From this match the identity of the pro tein may be determined Principle of Operation ProGest After the gel has been stained with silver Coomassie blue or fluorescent stains such as SYPRO Ruby Molecular Probes Oregon USA a gel sample no larger than 2mm in diameter and up to 2mm in depth is excised using either the Flexys Protein Picker or manually using a clean pipette tip or scalpel blade It is not necessary to mince the gel core into smaller pieces The gel pieces are placed in specially designed 96 well microtiter plates red colored that are mounted on the Reaction block within the ProGest These Reaction plates simply and effectively allow for the addition and removal of reagents without the liquid handling portion of the robot Dual Probes see diagram on page 4 coming into contact with the sample at any stage and thereby prevent ing cross contamination from one sample to another At the bottom of each well
69. position Screw 3 Pull the syringe plunger down into the threaded cou pling and tighten the 0 05 grub screw 4 Replace the clear acrylic cover and check the opera tion of the syringe See Priming the dilutor on page 96 Air bubbles cannot Screw couplings Turn the unit off be cleared from may be loose 2 Check the screw syringe during allowing air to couplings to the priming enter system reservoir bottle dilutor valves and syringes Note AII screw couplings should be fingertip tight Investigator ProGest 3 28 01 107 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 6 Troubleshooting Table 1 Problem Possible Cause Solution Rackis accidentally ProGest cover is The run must be moved during oper ation left open during run PAUSED and then CANCELLED The rack will move to the correct position when a new run is started Rack is accidentally moved during run and inner probes get pushed up inside the stainless steel outer probes An Autosampler error message and step number appears on the dis play ProGest cover is left open during run 1 Press OK to clear the error message and cancel the run or turn the unit off Before proceeding the probes must be correctly read justed See Probe Adjustment on page 94 Note The tray will be moved to the correct posi
70. r source for quality TecmoogyGroup new and certified used pre owned equipment FAST SHIPPING AND SERVICE CENTER REPAIRS WE BUY USED EQUIPMENT DELIVERY Experienced engineers and technicians on staff Sell your excess underutilized and idle used equipment TENS OF THOUSANDS OF at our full service in house repair center We also offer credit for buy backs and trade ins IN STOCK ITEMS www artisantg com WeBuyEquipment EQUIPMENT DEMOS HUNDREDS OF InstraV ea REMOTE INSPECTION LOOKING FOR MORE INFORMATION MANUFACTURERS Remotely inspect equipment before purchasing with Visit us on the web at www artisantg com 7 for more our interactive website at www instraview com 7 information on price quotations drivers technical LEASING MONTHLY specifications manuals and documentation RENTALS ITAR CERTIFIED yop aed Contact us 888 88 SOURCE sales artisantg com www artisantg com
71. raction Source Acetonitrile Vol 20u1 44 Extraction with CH3CN Waiting for timer 1 45 Shrinking with CH3CN Source Acetonitrile Vol 30u1 Hold Time 15 minutes 0 seconds 46 Purge wells Source Acetonitrile Vol Oul Hold Time 10 minutes 0 seconds 47 Rinse Inside 2000ul Outside 2000u1 48 Turn off heaters Heater OFF Investigator ProGest 3 28 01 41 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols Ag Destain Trypsin Digestion Destain met Step Number Description 1 Moving rack to run position Start X 2100 Y 925 Z 110 Stop X 447 Y 925 2 Moving rack to run position Start X0 Y 925 Z 110 Stop X 93 Y 925 3 Moving rack to run position Start X 300 Y 925 Z 80 Stop X 419 Y 925 4 Pierce Sheet Source Reservoir Vol Oul Hold Time 0 seconds 5 Turn off Heaters Heater OFF 6 Initial Rinse Inside 2000u1 Outside 3000u1 7 Destain Source Destain Vol 50ul Hold Time 15 minutes 0 seconds 8 Wash with Bicarbonate Source 25mM Bicarbonate Vol 100yl Hold Time 10 minutes 0 seconds 9 Wash with Bicarbonate Source 25mM Bicarbonate Vol 100u1 Hold Time 10 minutes 0 seconds 42 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols Step
72. ring standard operation Chapter 3 Protocols lists step by step each of the proto cols provided with the ProGest Chapter 4 Method Editor describes the Investigator ProGest Method Editor software provided with the ProGest which enables the user to create or edit digestion protocols network several ProGest instruments and run the system s by means of an attached PC Chapter 5 Maintenance describes some routine main tenance procedures along with cleaning and storage information for the system Chapter 6 Troubleshooting provides some basic trou bleshooting tips with possible causes and solutions Appendix A Trypsin Digestion Kit contains instruc tions for preparing the ProGest Trypsin Digestion Kit recommended for use with the Investigator ProGest Pro tein Digestion Station 12 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 1 Introduction Appendix B ProGest Validation Kit describes the kit available for use in the set up and validation of the instru ment Investigator ProGest 3 28 01 13 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 1 Introduction 14 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation Operation This chapter
73. safety do not touch the cables or other accessories while power is applied to these Always switch off the power before connecting or disconnecting the cables or accessories 5 Replace fuses with only the same type and rat ing Using an incorrect fuse could result in a fire hazard D Safety amp Operating Symbols The following symbols appearing on the unit or in the manual are defined as follows Warning The symbol of the stop sign is used through out the manual to indicate potential dangers that might result in personal injury or damage to property Caution The symbol of the crossed out bomb is used throughout the manual to indicate conditions that could cause damage to the unit Note This statement is used throughout the manual to highlight important information about the system Installation Warning lIf there is any evidence that the ProGest unit has been damaged in shipping do not plug the unit into the power line Contact Genomic Solutions Technical Service for advice 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Safety Precautions Power Requirements The ProGest uses a three prong power cord that includes a ground wire The unit must be connected to a properly grounded power supply outlet to ensure proper operation If there is any doubt about the intended power supply outlet a qualified
74. should be placed in or around the field of movement of either the horizontal or vertical arm of the robot e Warning You must not operate the robot without closing the protective cover as the moving arm and needle can lead to injuries The leakage current of this instrument is within the limits allowed by international safety standards for lab oratory equipment An efficient ground connection is imperative for the protection of the user Cleaning dismantling maintenance adjustment and repair should only be performed by personnel trained in such work and who are aware of the possible dan gers involved 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 5 Maintenance Warning Voltages present inside the instrument are potentially dangerous If there is a problem with the instrument make sure to switch the unit off The power cord should be removed until qualified service person nel have repaired it This is to prevent anyone from inadvertently using the instrument thus causing possi ble harm to themselves or damage to the instrument itself Genuine spare parts and consumables were specially designed for the instrument We must exclude any war ranty for damage resulting from modifications of the software modifications of the hardware operation deviating from the manual use of non genuine parts and cons
75. should only be operated with the protective cover closed in a well ventilated location Please consult your organization s safety regulations as to whether this instrument should be operated in a specially designated area or fume hood Please make sure that nothing interferes with the move ment of the instrument parts Nothing should be placed in or around the field of movement of either the hori zontal or vertical arm of the robot You must not oper ate the robot without closing the protective cover as the moving arm and probe can lead to injuries Voltages present inside the instrument are potentially dangerous If there is a problem with the instrument make sure to switch the unit off The power cord should be removed until qualified service personnel have repaired the unit This is to prevent anyone from inadvertently using the instrument thus causing possi ble harm to themselves or damage to the instrument itself Investigator ProGest 3 28 01 11 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 1 Introduction About the User s Manual This chapter provides system component information safety instructions a system overview and a description of the principles involved in the digestion procedure uti lized in the ProGest station There are five more chapters and an appendix in the man ual Chapter 2 Operation describes how to operate the ProGest du
76. stigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols Step Names Description 44 Extraction with CH3CN Waiting for timer 1 45 Shrinking with CH4CN Source Acetonitrile Vol 30u1 Hold Time 15 minutes 0 seconds 46 Purge wells Source Acetonitrile Vol Oul Hold Time 10 minutes 0 seconds 47 Rinse Inside 2000ul Outside 2000u1 48 Turn off heaters Heater OFF Investigator ProGest 3 28 01 51 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols 52 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization 4 Method Editor This chapter will take you through all of the functions and capabilities of the Investigator ProGest Method Editor Method Editor software In it you will learn how to create new protocols edit protocols network systems setup and run methods by means of a PC Method Editor Overview The Method Editor enables you to create and edit meth ods for the ProGest The Method Editor also has the abil ity to control several ProGest and ProMS instruments connected by cable to your PC This allows ease of method transfer by allowing you to transfer methods cre ated on your PC
77. tents vi 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Safety Precautions Warnings amp Safety Precautions The following precautions should be observed before using the Investigator ProGest Protein Digestion Sta tion ProGest and any associated apparatus Although units and accessories should normally be used with non hazardous voltages there are situations where hazardous conditions may be present This unit is intended for use by qualified personnel who recognize shock hazards and are familiar with the safety precautions required to avoid possible injury Read the Operator s Manual carefully before operating the ProGest Safety Warning 1 Maintain a well ventilated laboratory 2 Avoid a wet work area Always work with dry hands while standing on a dry insulated surface capable of withstanding the voltage being used The work area should be dry before using the unit while the power is on 3 Plug the instrument into a grounded power line Ensure that the power outlet in the labora tory is properly grounded Also inspect the power line cord and accessory cables before each use for any damage Investigator ProGest 3 28 01 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Safety Precautions 4 Beaware that high voltages could be a shock hazard For greatest
78. tep Properties Network Status The Menu and Tool Bar allows access to menus for loading and saving methods printing methods and accessing other parameters The Step List shows a list of all the steps in the method You can select a step by clicking the left mouse button on it At the bottom of the step window are two buttons controlling the simulator These allow the user to view the ProGest s movements prior to actu ally running the protocol The Step Properties once a step has been selected the step parameters will appear in this section Depending upon the step there can be several screens of parame ters You may adjust any of the step s parameters and the method is updated automatically but not saved To save any changes select the Save icon on the toolbar or select Save as from the File menu Investigator ProGest 3 28 01 55 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization The Network Status if you are using networking this part of the screen will show all of the Genomic Solu tion devices connected to your PC the instrument type and its status Menu Items and Tool Bar Listed below is a description of each of the software functions available in the menu and on the tool bar File The following functions are found under the File menu in the Method Editor software New This command allo
79. terior reagent area and reaction and collection areas with a damp cloth IV 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Safety Precautions Warning Be sure to power the instrument off and dis connect the power cord before cleaning the instrument DO NOT allow any liquid to enter the inside of the power unit This could damage the unit and present a potential shock hazard Investigator ProGest 3 28 01 V Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Safety Precautions Letter of Conformity CE GENOMIC e SOLUTIONS DECLARATION OF CONFORMITY Manufacturer s Name Genomic Solutions Inc Manufacturer s Address Genomic Solutions Ltd Unit 3 Forge Close Little End Road Eaton Socon St Neots Cambridgeshire United Kingdom Declares that the product Product Description Protein Digestion System Model Investigator ProGest Conforms with the protection requirements of Council Directive 89 336 EEC relating to electromagnetic compatibility By application of the following standards BS EN 61326 1 1997 Electrical Equipment for Measurement Control and Laboratory Use BS EN 60555 2 1995 Mains Harmonic Signature Andy iech John Parker EWE Opiratiiia Engineering Manager Genomic Solutions Ins Genomic Solutions Ltd Linited Sones United Kingdom
80. th CH3CN Source Acetonitrile Vol 50ul 11 Start Timer Starting Timer 1 Time 15 minutes 0 seconds 12 Neutralizing with ABC Source 50mM Bicarbonate Vol 50yl 13 Reaction Time Waiting for Timer 1 14 Shrinking gel Source Acetonitrile Vol 100u1 Hold Time 10 minutes 0 seconds 15 Set Heaters to High Heater HIGH 16 Reduction with DTT Source DTT Vol 30u1 Hold Time 10 minutes 0 seconds 17 Turn off Heaters Heater OFF 38 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols Step Number Description 18 Reduction cooling Delay 20 minutes 0 seconds 19 Alkylation Source Iodoacetamide Vol 30u1 Hold Time 15 minutes 0 seconds 20 Washing with ABC Source 50mM Bicarbonate Vol 40ul Hold Time 10 minutes 0 seconds 21 Washing with CH3CN Source Acetonitrile Vol 50u1 Hold Time 15 minutes 0 seconds 22 Shrinking with CH4CN Source Acetonitrile Vol 50u1 Hold Time 15 minutes 0 seconds 23 Purge wells Source Acetonitrile Vol Oul Hold Time 10 minutes 0 seconds 24 Prepare enzyme Source 25mM Bicarbonate Vol 950u1 Destination Trypsin Vol 900u1 25 Mixing enzyme Source Trypsin Vol 600u1 Destination Trypsin Vol 600u1 26 Mixing enzyme Source Trypsin Vol 600u1 Destination Trypsin Vol 600u1 Investigator ProGest 3 28 01 39 Artisan Tec
81. the edi tor Refer to Network Status on page 82 for more information remote control the Method Editor software allows the user to start stop or pause a ProGest You can also select sample wells directly from the PC by means of the Method Editor program Refer to Controlling ProGest Remotely on page 84 for more information General steps involved in the operation of the ProGest are as follows 6 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 1 Introduction Isolate the protein samples to be analyzed this step can be performed manually or auto matically by the Flexys Protein Picker Add the ProGest prepacked reagents or fill up the reagent and solvent tubes Prime the syringes Load the reaction block with a red colored Reaction plate containing the gel pieces Load the collection rack with the blue colored Collection plate Select the protocol to be run Select the wells in the red Reaction plate that contain gel pieces Close the cover Execute the program Method Protein Identification Enzymatic digestion is a standard technique used as part of the sample preparation process for a number of analyt ical techniques which can include peptide mapping by LC Liquid Chromatography and CE Capillary Electro phoresis but most commonly by mass spectrometry by either MALDI TOF or MS MS The profile of the
82. the front of the instrument to power the system off Investigator ProGest 3 28 01 35 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation 36 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols Protocols Listed in this chapter is a step by step description of the default digestion methods provided with the ProGest and also the Silver Destain Method Short Trypsin Digestion Default1 met Step Number Description Moving rack to run position Start X 2100 Y 925 Z 110 Stop X 447 Y 925 Moving rack to run position Start X0 Y 925 Z 110 Stop X 93 Y 925 Moving rack to run position Start X 300 Y 925 Z 80 Stop X 419 Y 925 Pierce Sheet Source Reservoir Vol Oul Hold Time 0 seconds Turn off Heaters Heater OFF Investigator ProGest 3 28 01 37 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols Step e Number Description 6 Initial Rinse Inside 2000u1 Outside 3000u1 7 Washing with Bicarbonate Source 25mM Bicarbonate Vol 100u1 Hold Time 0 seconds 8 Soak in ABC Delay 20 minutes 0 seconds 9 Purging ABC Source Acetonitrile Vol Oul Hold Time 0 seconds 10 Washing wi
83. the network view A menu list will appear Select Start Device A message will appear asking if you would like to enter a start delay f Yes enter the desired delay time in hours and minutes After the delay the ProGest will start to run the method that was last sent to it or selected on the instrument itself If No the ProGest will start to run the method that was last sent to it or selected on the instrument itself Investigator ProGest 3 28 01 85 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Stop or Pause ProGest Remotely To stop or pause the ProGest from the Method Editor Click the right mouse button on the device in the network view A menu list will appear Select Stop Device The ProGest will pause at the end of its current movement message will be displayed on the PC asking if you would like to Continue the run or Cancel it Select Continue to continue running the protocol Select Cancel to stop the protocol Select Sample Positions Remotely To change the active sample wells in the sample plate on the ProGest by the Method Editor Double click on the device in the network view A menu list will appear Choose Select Tubes A picture of a 96 well plate will appear The Method Editor will retain which wells were selected last You may select or de select pairs of wells by
84. tion when a new run is started 108 3 28 01 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Investigator ProGest Chapter 6 Troubleshooting Table 1 Problem Possible Cause Solution Reaction wells not emptied by nitro gen pressure 1 Nitrogen supply not turned on 2 Malfunction of nitrogen purge valve 3 Wrong reaction tray used 1 Check nitrogen supply at rear of unit 2 Make sure nitro gen is turned on See Check Nitro gen Valve on page 97 3 96 well trays must be red ones with small holes in the bottom Reaction wells do not drain com pletely 1 Sealing film not sealing around probe 2 Reaction plate is dirty 3 Nitrogen pres sure incorrectly set 1 Check that the sealing film was perforated cor rectly See Prepar ing the Reaction Plate on page 22 2 Make sure the reaction plate is new 3 Adjust nitrogen pressure to at least 15psi Ibar Investigator ProGest 3 28 01 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 109 Chapter 6 Troubleshooting Table 1 Problem Possible Cause Solution Strange values at the display of the temperature con troller 1 Heater cable not connected 2 Other malfunc tion 1 Firmly connect and secure the cable of the reac tion bl
85. tions in Chapter 2 Operation to run the system selecting the GSI Trypsin Digestion Protocol as the method of choice A 116 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Appendix B ProGest Validation Kit Appendix B ProGest Validation Kit The ProGest is a fully automated workstation designed for high throughput in gel protein digestion The ProGest validation kit provides a set of standard proteins to aid in the set up and validation of the instrument In addition to the ProGest Validation Kit the following reagents kits and equipment will be required ProGest Trypsin digestion kit Cat No 80 0210 Acetonitrile Vacuum microcentrifuge with a 96 well microplate rotor ProMS or MS Kit Contents The ProGest Validation Kit contains the following com ponents 96 well pink microplate containing approximately 1 5 picomoles of Coomassie blue stained protein standards in gel plugs The following standard proteins are included Soybean Trypsin Inhibitor Investigator ProGest 3 28 01 B 117 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Appendix B ProGest Validation Kit Carbonic Anhydrase Ovalbumin Glutamate Dehydrogenase Bovine Serum Albumin ProMS Matrix Solution e ProMS Peptide Matrix a cyano hydroxy cinnamic acid Instructions
86. to access the nitrogen valve through the ProGest software 1 Select Plate Configuration from the keypad 2 Select Setup from the keypad The Engineering Options window appears 3 Select Nitrogen from the keypad to turn the valve on You should be able to hear the valve click and hear the hiss of the nitrogen coming through the stainless steel probes A further Investigator ProGest 3 28 01 97 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 5 Maintenance check would be to undo the probe assembly from the robot arm using the large finger screw and to dip the end of the probes into a container of water to see if the nitrogen is bubbling through both outer probes If Nitrogen appears to be coming through the probes select Nitrogen again to turn the valve off If there is no hissing noise or no apparent bub bling of Nitrogen through the water press Nitro gen again to turn the valve off Make sure the ProGest is connected to a compressed nitrogen tank and that the tank is not empty If the problem persists contact Genomic Solu tions Technical Service for assistance Check Heater The parameters of the temperature controller have been carefully set at the factory but it may be necessary to check the function 1 2 Select Plate Configuration from the keypad Select Setup from the keypad The Engineering Options window appears Select Heater
87. to be added to each sample well from the drop down menu list Enter the desired amount of reagent in u1 to be added Enter the amount of hold time in hours min utes or seconds Select the desired aspiration and dispense time in ml min default speed 1Oml min Select an airgap volume in ul if desired Select an equalization time in seconds Select a discard volume in pl Select a droplet aspiration in ul 3 28 01 73 Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Purge Enter the amount of time for the nitrogen purge in seconds Note The needles move to a purge position inside the pair of sample wells and the nitrogen valve is opened to allow the liquid in the wells to be purged into waste Enter the amount of post purge time in sec onds This is a delay after the nitrogen is turned off to allow the nitrogen pressure to decay This is normally set to zero Rinse Enter the amount of inside wash volume in ul Default is 1000u1 Enter the amount of outside wash volume in ul Default is 1000u1 74 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Dilutor Select the desired aspiration and dispense time in ml min Default speed 10ml min e S
88. tor ProGest 3 28 01 57 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Open This command allows the user to open an existing proto col from the protocol directory provided with the ProGest 1 Select Open from the File menu The Open Editor window will appear bun rens a mb er fs mu LP EB sb hires ir Ertl bres rent E Eri Fer rua THE NL d nen bast ret aj 1 Ir Pinna ma fimul pps Dagestan bl ethene tt 1 2 Select highlight the desired protocol and select Open to bring the protocol into the ProGest Diges tion Method Editor window 3 The protocol will appear in the left hand side of the window where it can be edited or amended as desired Save This command allows the user to select a directory and filename in which to save the opened protocol after mak ing changes to it 58 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization 1 Select Save from the File menu The Save As win dow appears in which you can name a directory and file in which to save the protocol as a digestion method met Save As This command allows the user to select a directory and filename in which to rename the opened protocol or save an opened protocol not previous
89. trumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 6 Troubleshooting Table 1 Problem Possible Cause Solution At the display this message appears Problem with Autosampler fol lowed by some softwarecommands and the last com mand that was issued Z drive error X drive error or Y drive error This happens if something hinders the movement of the vertical arm Z drive of the robot or something hin ders the movement of the horizontal arm X Y drive 1 Check the work area 2 Check the align ment and orienta tion of the rack 3 If the inner probes have been pushed up inside the stainless steel outer ones they need to be reset See Probe Adjust ment on page 94 4 If the probe is damaged replace it Note Always use original consum ables and spare parts supplied by Genomic Solutions 106 3 28 01 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Investigator ProGest Chapter 6 Troubleshooting Table 1 Problem Possible Cause Solution Dilutor syringe Syringe plunger To reset the fails to prime at the may have become syringe beginning of arun detached from the Turn the unit off or dispense liquid threaded coupling 2 Unscrew the four correctly during a The plunger is screws holding the run held in place by a clear acrylic cover single 0 05 grub in
90. umables Installation Installation of the system is carried out by Genomic Solu tions personnel or their representative Please do not try to install the system yourself Changing the dilutor syringe If the syringe is damaged or does not work properly an exchange is required Please replace the syringe with the correct Genomic Solutions part Note Make sure that the piston operating rod is NOT in the uppermost position Use the software to lower the Investigator ProGest 3 28 01 91 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 5 Maintenance piston operating rod as follows This allows you to dis mount and mount the syringe without risk of damaging the rod or syringe 1 Switch on the instrument and press Plate Con figuration at the keypad 2 Select Setup from the keypad 3 Press the key Load Syringe The piston operat ing rod moves to the lower position The instru ment screen changes then changes to prompt the user 4 Unscrew the four screws holding the plexiglass syringe assembly cover 92 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 5 Maintenance Syringe and valve assembly 5 Investigator ProGest Unscrew the mounting screw at the bottom of the piston operating rod Unscrew the syringe from the valve using the knurled
91. urce Trypsin Vol 600ul Destination Trypsin Vol 600u1 28 Mixing enzyme Source Trypsin Vol 600ul Destination Trypsin Vol 600u1 44 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols Step N mbr Description 29 Mixing enzyme Source Trypsin Vol 600u1 Destination Trypsin Vol 600u1 30 Adding Enzyme to samples Source Trypsin Vol 15ul Hold time 10 minutes 0 seconds 31 Moving rack for extraction Start X 400 Y 925 Z 80 Stop X 1642 Y 925 32 Moving rack for extraction Start X 1610 Y 925 Z 80 Stop X 1642 Y 925 33 Set Heaters to 37 C Heater LOW 34 Digestion first stage Delay 1 hour 30 minutes 0 seconds 35 Rinse needles Inside 1000u1 Outside 1000u1 36 Adding water to gel Source Water Vol 10pl 37 Digestion second stage Delay 6 hour 0 minutes 0 seconds 38 Rinse needles Inside 2000u1 Outside 2000u1 39 Adding ABC Source 25mM Bicarbonate Vol 10ul 40 Extracting peptides Delay 10 minutes 0 seconds Investigator ProGest 3 28 01 45 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 3 Protocols Step i ds Number Description 4 Adding CH3CN for extraction Source Acetonitrile Vol 20u1 42 Incubation with CH3CN Delay 10
92. white block Carefully readjust the position of the blue fer rule on the inner probe and tube as shown 94 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 5 Maintenance Tubing Locking Screw Ferrule Inner Probe Push the probe back into the assembly and tighten the locking screw in the white block The correct protruded length of the inner probe from the outer stainless steel probe is 8 0 mm stainless Steel Probe 8 0 mm Inner Frobe Investigator ProGest 3 28 01 95 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 5 Maintenance Priming the dilutor If the system has not been used for some time it may be necessary to fill the syringe and the tubing leading to the reservoir with reservoir fluid and purge air It is also good practice to prime the syringe before each run l 2 3 4 Select Plate Configuration from the keypad Select Setup from the keypad Press Prime Dilutor to start priming Press Stop Dilutor after about 10 strokes to can cel priming Check for bubbles in the syringe or tubing Con tinue priming until no bubbles are seen Upgrading Software If a new version of software needs to be loaded into the ProGest instrument it can be loaded manually by insert ing a floppy disk with the software downloaded
93. ws the user to create a new protocol document by selecting steps adding or deleting steps and also to name and save the resulting protocol method Select New from the File menu A window will appear that allows the user to select which type of method to cre ate 56 3 28 01 Investigator ProGest Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 4 ProGest Method Editor Customization Cire ura TELE Treba minh ups cd erha pou eant Hi CRE 1 Select ProGest The Method Editor window remains open 2 Tocreate a protocol select Append Step from the Edit menu The New Method Step window appears 3 Select the appropriate step from the drop down menu list Select OK 4 The step will appear in the left half of the Method Editor window as Step 1 Enter the required infor mation for the selected step Refer to List of Steps on page 65 for information about each step option 5 Continue appending steps as desired Note You may also insert steps or delete steps under the Edit menu nsert step will insert a new step at the selected or highlighted step Delete step will delete the selected or highlighted step Refer to Insert Step and Delete Step on page 61 for more infor mation 6 When the protocol is complete you may select to name it by selecting Set Method Title from the File menu or save it by selecting Save As from the File menu Investiga
94. ww artisantg com Chapter 2 Operation 2 The start of the run can be delayed up to 23 hours and 50 minutes This will enable the user to time the digestion to end whenever desired 3 Select the delay time in hours and minutes if desired then press Start You may also select to Cancel the run from this screen This will take you back to the Main menu 4 During the run various messages will be dis played describing the status of the system at that time an approximate estimation of completion time and or any error conditions that may arise 5 When the run is finished the screen will indicate that the program is finished Interrupting Aborting the program The program can be stopped or interrupted at any time during its operation by the following steps 1 Select Pause Digestion to temporarily stop the program The system will finish the current oper Investigator ProGest 3 28 01 31 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chapter 2 Operation ation and then stop A window will appear asking for instructions 2 Press Continue to continue the run or Cancel to abort the run If you select Cancel the pipetting arm and dilutor will reset and a window will appear informing you that the run has been can celled lt GENOMIC _ 2 SOLUTIONS 3 Select Return to Main menu The system will go back to the Main menu 32 3 28 01 Investigator Pr

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