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Proteomics of Medicago truncatula

1. do not use the computer to do other work such as checking email writing paper or searching the internet as we have found that these activities can interfere with the instrument s communications and efficiency One hour after all samples are acquired the system automatically goes idle to stop the Ultimate 20 The sizes of LC MS MS data files are large 15 25 MB and they can take up the hard drive space quickly Move your data files to the common network drives CDs or DVDs for long term storage Proteomics page 48 of 62 Medicago truncatula Handbook version March 2007 5 3 Database searching A public Mascot server can be assessed through the Matrix Science website http www matrixscience com search form select html This web based public server is free and allows one to upload mass spectra data files and perform the database searches on line However there are some limitations on the web based Mascot server a maximum of 2 simultaneous searches per user b maximum file upload 5 Mb note our LC MS MS data are typically in the range of 15 25 MB c maximum of 300 spectra in a single MS MS search and d custom databases can not be searched In addition users are requested not to submit more than one very large search at a time as the search can take a long time Very large search refers to the search in which no enzyme specificity is given or more than 5 variable modifications of amino acids are used or the search is agains
2. 7 Abbreviations 1D 2 DE 2D LC AA AM amu APS ASB14 BSA C terminal C M CBB G 250 cc CHAPS cm cps DIGE DMSO dpi DTT EDTA ESI EST fm g g H20 HPLC hrs IAA ICAT IDA IEF IPG iTRAQ L LC MS MS M m z mA MALDI TOF MS MB mg M gCl ml mm Proteomics version March 2007 One dimensional Two dimensional electrophoresis Two dimensional liquid chromatography Amino acid arbuscular mycorrhizal Atomic mass unit Ammonium persulphate amidosulfobetaine 14 Bovine serum albumin Carboxy terminal chloroform methanol Commassie Blue G 250 Cubic centimeters 3 3 Cholamidopropyl dimethylammonio 1 proanesulfonate centimeter Counts per second Difference gel electrophoresis dimethylsulfoxyde Dots per inch Dithiothreitol ethylene diaminetetraacetic acid Electrospray ionization Expressed sequence tags Femto moles Gram Gravity Water High performance liquid chromatography Hours Iodoacetamide Isotope coded affinity tagging Information dependent acquisition Isoelectric focusing immobilised pH gradient gel Isobaric tags for relative and absolute quantification Liter Liquid chromatography mass spectrometry mass spectrometry Molar Mass charge Milli amperes Matrix assisted laser desorption ionization time of flight mass spectrometry Mega bytes Milli gram Magnesium chloride Milli liter Milli meter page 53 of 62 Medicago truncatula Handbook mM Mohm
3. C 13 Mix carefully and precipitate nucleic acids overnight at 20 C D Second day 1 Centrifuge at 12000 g for 35 min at 4 C 2 Throw away the supernatant and suspend the pellet in 200 ul DEPC water 3 Shake the tubes up side down and leave on ice 5 min 4 Add 1 volume of LiCL 4M to precipitate the RNA mix carefully and leave 4 5 hours in a cold room 4 C Centrifuge 12000 g 35 min at 4 C 6 Throw away the supernatant and suspend the pellet in 200 ul DEPC water m Proteomics page 20 of 62 Medicago truncatula Handbook version March 2007 7 Shake the tubes and leave on ice for 10 min 8 Precipitate the RNA overnight at 20 C with 0 1 volume sodium acetate 3M and 3 volumes of cold ethanol 96 kept at 20 C Third day 1 Centrifuge at 12000 g for 35 min at 4 C 2 Throw away the supernatant and re suspend the pellet in 200 ul of cold ethanol 75 kept at 20 C 3 Centrifuge at 12000 g for 35 min at 4 C 4 Dry the pellet about 1h and suspend in 50 ul of DEPC water leave about half a day and mix from time to time 5 Leave overnight at 20 C may also be kept at 80 C Fourth day Check the RNA quality on an agarose gel before its quantification with a spectrophotometer Sambrook and Russell 2001 Protein extraction 1 Pool all the phenolic phases b 1 to 3 and complete with phenol up to 10ml 2 Homogenize in 10 ml of extraction buffer Table 3 and proceed as previously described see para
4. currently have MSDB NCBInr dbEST Swiss prot and several custom local databases The legume specific protein database is named legprot which was compiled mostly from Medicago truncatula 3 Select ABI MDS Sciex Analyst WIFF File in the Data Import Filter tab step4 If data file format rather than WIFF format is selected for QSTAR data files the search will fail 4 Add mass spectral data files you want to search into the Data file list window by clicking the Add File button step 5 Browse and select files and click Ok to add them into the search list 5 Click Run button to begin the search step 6 While searching 1s in progress do not use the computer for other work 6 When the search is finished click Status to view and print the search results The results can be opened by clicking on individual search results 6 Concluding remarks Proteomics is a fast changing field and new protocols are being established frequently Most procedures depend on high quality ingredients and careful experimentation to achieve good quality gels Especially procedures for pure protein samples are important for all subsequent steps and will require optimization in any individual lab So far the diversity of proteins 1n an organism make it impossible to visualize all proteins at once Compared to transcriptome analysis which can reliably track the expression of tens of thousands of genes proteomics has been limited in the number of pr
5. 1 add 1 5ml of the prepared protein solution 0 7mg ml to the equilibrated matrix 2 allow phosphorylated proteins to bind to the matrix by shaking rotator at 4 C for 30min 3 remove unbound protein flow through FT with a centrifugation step 13 350g 4 C 2min as supernatant 4 save the flow trough to monitor the success of the enrichment Washing of unbound protein 1 add 1 5mL PEB to the loaded matrix 2 do not vortex 3 mix manually by flipping your fingertip against the bottom of the tube until the matrix 1s dispersed 2 3 times 4 centrifuge at 13 350g and RT for 2min 5 wash unspecifically bound proteins for a total number of five washing steps Elution 1 elute using 800uL freshly prepared Phosphoprotein Elution Buffer Table 15 by shaking for 30min RT 2 centrifuge at 13 350g and RT for 2 min 3 transfer the supernatant to a Schott GL14 glass test tube Cat number 23 175 11 59 Table 15 Phosphoprotein Elution Buffer PEB 8M urea 500mM potassium pyrophosphate Adjustment to the pH of 9 0 with H3PO Protein Precipitation Wessel and Flugge 1984 Use 50uL of the retained sample solution 100uL of the flow through and the complete eluate of 800uL for the precipitation General protocol 1 add 4 volumes of Methanol and mix until homogeneity of turbidity 2 add 1 volume of chloroform and mix until homogeneity of turbidity 3 add 3 volumes of ddH2O and mix until homogeneity of turbidity 4 spin f
6. MP mTorr Mud PIT N terminal N2 NA ng nl nm NTES buffer PAG PMF PMSF psi Q TOF rpm SDS TBP TCA TEMED TFA Tris MES U V yr Hg ul Proteomics version March 2007 Milli molar Mega ohm Multiplexed proteomics Milli torr Multidimensional protein identification technology Amino terminal Nitrogen Nuclei acid Nano gram Nano liter nanometer NaCl Tris HCI EDTA SDS buffer Plant and animal genome conference Peptide mass fingerprinting Phenylmethylsulphonylfluoride Pounds per square inch Quadropole TOF Revolutions per minute sodium dodecylsulphate tributyl phosphine Trichloroacetic acid N N N N Tetramethylethylenediamine Trifluoroacetic acid Tris morpholinoethanesulfonic acid Units Voltage Year Micro gram Micro liter page 54 of 62 Medicago truncatula Handbook version March 2007 7 References Agrawal G K M Yonekura Y Iwahashi H Iwahashi and R Rakwal 2005a System trends and perspectives of proteomics in dicot plants Part I Technologies in proteome establishment J Chromatogr B Analyt Technol Biomed Life Sci 815 109 23 Agrawal G K M Yonekura Y Iwahashi H Iwahashi and R Rakwal 2005b System trends and perspectives of proteomics in dicot plants Part II Proteomes of the complex developmental stages J Chromatogr B Analyt Technol Biomed Life Sci 815 125 36 Agrawal G K M Yonekura Y Iwahashi H Iwahashi and R Rakwal 2005c System trends and pers
7. and quantitation Curr Opin Chem Biol 6 46 50 Maltman D J W J Simon C H Wheeler M J Dunn R Wait A R Slabas 2002 Proteomic analysis of the endoplasmic reticulum from developing and germinating seed of castor Ricinus communis Electrophoresis 23 626 39 Mathesius U Imin N Natera S H A and Rolfe B G 2003 Proteomics as a functional genomics tool In Plant Functional Genomics Methods and Protocols Methods in Molecular Biology Series Vol 236 Ed E Grotewold Humana Press New Jersey 395 413 Mathesius U G Keijzers S H Natera J J Weinman M A Djordjevic and B G Rolfe 2001 Establishment of a root proteome reference map for the model legume Medicago truncatula using the expressed sequence tag database for peptide mass fingerprinting Proteomics 1 1424 40 Ni W V Sewalt K L Korth J W Blount G M Ballance and R A Dixon 1996 Stress responses in alfalfa XXI activation of caffeic acid 3 o methyltransferase and caffeoyl coenzyme A 3 O methyltransferase genes does not contribute to changes in metabolite accumulation in elicitor treated cell suspension cultures Plant Physiol 112 717 726 Ong S E and A Pandey 2001 An evaluation of the use of two dimensional gel electrophoresis in proteomics Biomol Eng 18 195 205 Otvos K T P Pasternak P Miskolezi M Domoki D Dorjgotov A Szcs S Bottka D Dudits and A Feher 2005 Nitric oxide is required for and promotes auxin media
8. e e e Number of Spots cn e 1 1 0 Log Yolume Ratio Figure 9 Distribution of spots from a DIGE experiment and correction of spot volumes to a normal distribution to allow statistical analysis by ANOVA The log volume ratio is the ratio of spot abundance between test and control Green spots are not significantly different in the two samples red spots are down regulated in one of the samples and blue spots are up regulated in the same sample compared to the control Figure 10 Matching of spots between gels All green circled spots are detected and matched between the gels Red spots are down regulated in one of the gels blue ones up regulated Any spot that is clicked by the mouse will have a purple circle Proteomics page 39 of 62 Medicago truncatula Handbook version March 2007 Figure 11 Differential display of spot volumes between two gels The purple spot is marked on the gel by clicking with the mouse and will be displayed as a Mountain Plot below the red marked spots are differentially displayed between the gels the green ones are similar fF DeCyder BVA batch 4 and 24 hr bva File Edit View Process Help OSM smear E PP Graph View Master No 3403 Standardized Log amp bundance Flagellin 4 hr 2 ANOVA Conditi Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirme
9. have been widely used to study biochemistry of Medicago Baier et al 1999 Daniell and Edwards 1995 Edwards and Dixon 1991 Gana et al 1998 Guo et al 1994 Ni et al 1996 Otvos et al 2005 Shorrosh et al 1994 Steward et al 1999 Suzuki et al 2005 Cell culture systems are often used in experiments that require large quantities of a homogeneous cell type that can be subjected to various conditions and multiple samples can be obtained from these conditions Experiments like this have been performed in the author s lab Lei et al Nagaraj et al Manuscripts in preparation A representative 2 DE gel of cell suspension culture 1s shown in Figure 1 pI 3 l pI 10 l A onm Lo E pe uat Ld Em 9m p E Figure 1 Representative 2 DE gel illustrating the resolution of M truncatula proteins extracted from liquid cell suspension cultures The sample was collected following treatment with a biotic elicitor The harvested sample was homogenized proteins were extracted and separated by 2 DE Molecular weight markers are present on the left of the 2 DE and are composed of MARK 12 unstained standard from Invitrogen 1 Grind approximately five grams of frozen cell tissue in a mortar pre chilled with liquid nitrogen i Add liquid nitrogen grind slowly as it boils off and then grind faster when the tissue is wet with liquid nitrogen Repeat the grinding step for a total of three tim
10. in 200 ul of ImM HCI so that final concentration 1s between 100 125 ng ul 8 Prepare working trypsin solution by diluting stock solution 1 to 10 with 25 mM ammonium bicarbonate 1 acetonitrile such that final concentration 10 to 12 5 ng ul Note the pH difference in stock solution 2 9 versus working solution pH 8 0 Trypsin activity is pH dependent At low pH trypsin has low activity and can be stored for approximately 2 weeks Trypsin working solutions must be used shortly after increasing pH to 8 1 e 2 hrs If not autolytic digestion products are observed at higher levels than desirable 9 Rehydrate dried gel plug bands in approximately 10 ul trypsin for 20 minutes on ice This allows the trypsin to infuse into the gel plug for in gel digestion 10 Remove excess trypsin solution by washing twice with 25mM ammonium bicarbonate to remove excess trypsin and to lower the autolytic peak abundances Proteomics page 42 of 62 Medicago truncatula Handbook version March 2007 11 Add 10 to 20 ul of 25 mM ammonium bicarbonate to sample vial to ensure proper hydration of the gel plugs bands during digestion at elevated temperatures 12 Digest at 37 C for 4 to 6 hrs Overnight digestions are possible but yield higher levels of autolytic trypsin products which may decrease sensitivity for low level protein digests Overnight digestions should only be considered for intense Coomassie stained gel plugs bands 13 Stop digestio
11. last step in which proteins need to be solubilised in a buffer not containing any DTT and ampholytes The recommended buffer contains 30 mM Tris 7M urea 2M thiourea and 4 w v CHAPS After solubilizing the proteins in this buffer the pH needs to be adjusted to between 8 9 ideally 8 5 to allow the labeling reaction to occur The pH can be adjusted with HCl or NaOH depending on pH of the sample The easiest thing is to spot a tiny drop of sample onto some pH paper Measure protein concentration of the samples and set aside 100 ug protein from each sample in a separate Eppi tube and mix an additional 50 ug of each sample into a separate tube for the internal control Adjust volumes of the sample with solubilization buffer to make them approximately equal To label the proteins the CyDyes first need to be diluted into anhydrous fresh dimethylformamide add 5 uL DMF to each tube this will give a 1 mM solution Keep dyes dark and at 20 C at all times until use Once the DMF is added the dyes will keep for 3 months at 20 C We found that a ratio of 200 pmol of dye to 100 ug protein 1s optimal After adding the appropriate dye to each sample put tubes on ice for 1 h in the dark Add 1 uL of 10 mM lysine to stop the reaction Leave on ice for 10 min Samples are ready to be mixed together and loaded Setup for gel loading Proteomics page 37 of 62 Medicago truncatula Handbook version March 2007 A typical setup of a DIGE experim
12. possible to characterize relatively large and labile biomolecules including peptides and proteins using MS MS based protein identification typically involves two different steps 1 analysis of peptides generated by proteolyic digestion and 2 database searching Digestion of protein can be achieved using various proteases but the most popular one has been trypsin which hydrolyzes the C terminal side of lysine and arginine unless the subsequent amino acid in the sequence is a proline Peptides produced by tryptic digestion normally possess two basic sites i e the basic lysine or arginine residual at the C terminal and the basic amine group at the N terminal This allows peptides to be protonated efficiently and hence enhances ionization of mass spectral sensitivity Multiple protonation sites also result in the production of multiple charged species in ESI although not readily in MALDI due to fundamental differences in the energetics of these two techniques The multiple charges render one important advantage 1 e the peptides can be easily distinguished from impurities which are typically singly charged This is especially useful when peptides need to be selected for tandem mass spectrometry MS MS analysis Currently the authors use an ABI QSTAR pulsar Q TOF for protein identifications This protocol describes the use of the QSTAR Q TOFMS coupled to a nano liquid chromatography system LC packings to analyze peptide mixtures generated by
13. red in test result is encountered you can then click Apply to begin compiling the new local database b Database Search MASCOT is a probability based search engine that uses mass spectrometry data to identify proteins by searching sequence databases Perkins et al 1999 It can perform peptide mass fingerprinting PMF sequence query and MS MS ion searches MASCOT Daemon is an automation script that allows one to query multiple data files through a batch search 1 To use the Mascot Daemon go to Programs and select Mascot Daemon It opens the Mascot Daemon menu as shown in Figure 17 AB IMOS Sciex Analyst WiFF He Schedule Drag and drap files into the area below Start now or click on Add Files C Starttat 3 44 37 AM Xe E August 2004 Realtime monitor Follow up Actions Auto print results External processes Follow up No follow up required fo P Wieeard results Repeat ab ratenvals of zi days zi Debe Came Files Pass data to E quM Figure 17 Steps for setting up Mascot Daemon The protocol describing the initial steps to setup a Mascot search is shown and the individual steps are circled and indicated by numbered arrows Proteomics page 50 of 62 Medicago truncatula Handbook version March 2007 2 First click task editor step 1 to open the search menu Enter the name of the task step 2 select the database which you want to search step3 We
14. sample should migrate to exactly the same spot To distinguish the proteins from the two samples they are labeled before the 1D separation by different fluorescent dyes Cy3 and Cy5 similar to labeling RNA ina microarray The intensity of fluorescence at each of the wavelengths for Cy3 and Cy5 is measured and the ratio gives an indication of the abundance of protein X from M or WT within gel matching Like in any other biological experiment one needs to run biological repeats of samples 1 e separately grown biological material separately extracted and separated on different 2D gels Each pair of repeats of M vs WT would be run on a separate gel There will therefore still be gel to gel variation that makes matching between gels difficult To improve between gel matching each gel also contains a third sample This is a mixture of all samples of the whole experiment e g M and WT of each biological repeat This mixture 1s labeled with a third dye Cy2 and run on each gel Therefore this internal control should be representing the same proteins on each gel and can be used to compare protein patterns between gels It also serves as an internal control for the statistical analysis of the protein abundance difference between the different samples The CyDyes have an NHS ester group which reacts with the epsilon amino group of lysine residues of proteins The dyes are called minimal dyes because only enough dye is used to labe
15. times the volume of the pellet more is also fine Flick the pellet or stir briefly with a clean glass rod to wash the TCA out Keep on dry ice for at least 30 min again this step can be extended to over night if the experiment needs to be interrupted Centrifuge as in 9 Remove the supernatant Repeat steps 10 and 11 After removing the second volume of acetone dry the pellet Either leave the tubes open on the bench at room temperature 1s fine at this stage as the TCA treatment should have inhibited proteases for at least 1 hr Alternatively dry the pellet for no more than 5 min in a rotary evaporator on low heat setting or leave tube open in the Proteomics page 12 of 62 Medicago truncatula Handbook version March 2007 15 16 17 freezer over night The acetone should evaporate Avoid over drying the pellet as it will be difficult to resuspend in the next step If you used an ultracentrifuge so far transfer the pellet into an Eppendorf tube otherwise leave in Eppendorf tube Suspend the pellet in a small volume of Solubilisation Buffer see Table 2 The volume depends on the size of the pellet We usually start with a volume slightly bigger than the pellet size The sample should be as concentrated as possible It will look like this mixture will stay almost dry but after rigorous vortexing several minutes and sonication in a sonic bath add some ice to the water the pellet will solubilise To help resuspension th
16. 08 formic acid using helium for 10 minutes Pressure should not exceed 20 psi Use only HPLC grade solvents 2 Fill pump heads with 50 isopropanol using a 20cc syringe This will dissolve salts that may deposit on the pump heads Proteomics page 45 of 62 Medicago truncatula Handbook version March 2007 3 Open the drain valve and purge pumps by pressing the PURGE button First purge pump A and then pump B on the Ultimate until no air bubbles come out Close the drain valve The Switchos also needs to be purged 4 Inspect the injection syringe for air bubbles Air bubbles of accumulate after long idle periods of time Remove air bubbles by pressing wash button on the panel of the autosampler If air bubbles still exit take the syringe out and manually remove the bubbles Incorrect sample volume may result if there are air bubbles in the syringe and the tubing 5 Check to make sure the trap column is in load position or the sample will be transported directly to the waste bottle If it is not in load position use the switch at the back of the Switchos to adjust the trap position 6 Check all waste bottles one for Switchos and two for Ultimate to avoid overflow during the run especially during the weekend 7 Turn on all pumps and check trap column backpressure Under the current setup the normal back pressure reading for trap column is about 85 psi 8 Set autosampler Famos to serial mode so that the Analyst software can c
17. 1976 with the modifications suggested by Ramagli and Proteomics page 14 of 62 Medicago truncatula Handbook version March 2007 Rodriguez 1985 This allows protein quantification even in samples containing components that usually interfere with the reactive of Bradford s assay Table 3 Extraction buffer 100ml for total and soluble proteins Table 4 Solubilization buffer for soluble Tris 0 5 M 6 055 g proteins Deionized water sqf 50 ml Sucrose 0 7 M 23 9 g Urea 9 M 5 4g KCl 0 1M 0 75g CHAPS 4 w v 0 4 g Thiourea 10mM 0 076 g Triton X 100 0 596 v v 50 ul EDTA 5mM 50 mM 10 ml DTT 20 mM 0 154 g B mercaptoethanol 2 2 ml IPG buffer 3 10 1 2 w v 200 ul PMSF 2 mM in DMSO 18 mg 1 ml DMSO MilliQ water sqf 10 ml MilliQ water sqf 100 ml can be stored at 20 C To be added just before using the solution Proteomics page 15 of 62 Medicago truncatula Handbook version March 2007 2 6 Extraction and solubilization of M truncatula root membrane proteins by chloroform methanol Valot et al 2004 and 2005 adapted from Ferro et al 2000 Sub cellular proteomics defined as the analysis of the expressed proteins of purified individual cell compartments has emerged as an interesting tool to complement total proteins data Jung et al 2000 In the case of biological membranes various studies were directed to identify membrane proteins of either microsomes Prime et al 2000 or plasma membranes Santoni et al 1998 amp 19
18. 2007 Analyst QS TOF MS Experiment 1 10 423 to 11 035 min from calibration wiff Oo 8 x m File Edit View Tools Explore Window Script Help e x la oi amp 5 Ba Ce Explore Mode la amp zhentian Lei IC x T HF JAS res t amp X JG UM us ARAS XA 2E Ade UHBS RELEASE ve IS IE OE R9 aa TOF MS Experiment 1 10 423 to 11 035 min from calibration wiff Max 243 9 counts od Configure a 3 56895839 164267 130e 004 10 4 08257345105359450e 001 R Security Configuration 705 2287 cul Hardware Configuration Report Template E ditor I Tune 1 f Resolution Optimization A Quantitative Optimization iE Manual Tuning ey TOF Mass Calibration 786 3398 z Acquire TE Build Acquisition Method Build Acquisition Batch zz Express View Explore 1 a Open Data File Gg Open Compound Database LZ Quantitate WC Quantitation Wizard Review Results T able e t a eo a c m E 786 8422 787 3427 787 8468 785 3336 784 8292 tst au es N78 5402 7880209 nce 7844 7846 7848 7850 7852 7854 7856 7858 7860 786 2 7864 7866 7868 7870 7872 7874 7876 7878 7880 7882 788 4 m z amu For Help press F1 EA lide W Ready IH ue start c e ah ao Z Analyst Q5 T0F M E 11 rtf WordPad 3D UY 407m Figure 14 Mass spectrum peak selection for TOF MS calibration Screen shot showing the selection of a peak for calibration of the TOF MS The d
19. 4397 Proteomics page 59 of 62
20. 99 of A thaliana germinating seed endoplasmic reticulum Maltman et al 2002 as well as of peribacteroid membranes from root nodules Saalbach et al 2002 Wienkoop and Saalbach 2003 As strictly hydrophobic proteins are not yet amenable to 2 DE analysis Seigneurin Berny et al 1999 we have developed a protocol to gain access to proteins associated with total membrane structures of M truncatula roots 1e microsomes This was achieved by combining a sub cellular partitioning process to 2 DE separation Valot et al 2004 and that has recently been validated for the study of the late stages of arbuscular symbiosis Valot et al 2005 1 Sample roots at least 10g of fresh material or of material stored at 80 C 2 All steps are carried out in a cold room 3 Crush with liquid nitrogen and add the extraction buffer E Table 5 2 ml of E buffer g of fresh material 4 Cell walls nuclei and most of the mitochondria will be precipitated by two successive steps of centrifugation 20 min at 12000 g and 16000 g Figure 4A 5 The supernatant corresponds to the total protein fraction from which 2 ml will be reserved for a subsequent phenolic extraction 6 The remaining volume is centrifuged one hour at 100000 g The second supernatant corresponds to the cytosolic fraction that will be like the total fraction submitted later to a phenolic extraction Figure 4A 7 The pellet containing the whole cell membranes correspon
21. 999 Correlation between protein and mRNA abundance in yeast Mol Cell Biol 19 1720 30 Hamdan M and P G Righetti 2002 Modern strategies for protein quantification in proteome analysis advantages and limitations Mass Spectrom Rev 21 287 302 Hirano H N Islam and H Kawasaki 2004 Technical aspects of functional proteomics in plants Phytochemistry 65 1487 1498 Imin N F De Jong U Mathesius G van Noorden N A Saeed X D Wang R J Rose and B G Rolfe 2004 Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts Proteomics 4 1883 96 Karp N A D P Kreil and K S Lilley 2004 Determining a significant change in protein expression with DeCyder during a pair wise comparison using two dimensional difference gel electrophoresis Proteomics 4 1421 1432 Lei Z A M Elmer B S Watson R A Dixon P J Mendes and L W Sumner 2005 A two dimensional electrophoresis proteomic reference map and systematic identification of 1367 proteins from a cell suspension culture of the model legume Medicago truncatula Mol Cell Proteomics 4 1812 25 Le Signor C K Gallardo J M Prosperi C Salon L Quillien R Thompson and G Duc 2005 Genetic diversity for seed protein composition in Medicago truncatula Plant Genetic Resources 3 59 71 Lilley K S A Razzaq and P Dupree 2002 Two dimensional gel electrophoresis recent advances in sample preparation detection
22. Ag which is visible as a dark brown to black spot on the gel Silver does not uniformely bind to all proteins some proteins are not stained at all page 30 of 62 Medicago truncatula Handbook version March 2007 with silver and appear as negative spots on the clear even clearer than the gel background and typically different spots on a silver stained gel have slightly different shades of brown While comparing the same protein across several gels is semiquantitative it is not a reliable method for quantitative proteomics Silver staining 1s useful for staining proteins sensitively to optimize 2D separation and gel running Prepare all solutions just before use or extemporaneously and with highest grade chemicals all in ultrapure water Solutions 1 Fixation solution 10 v v acetic acid 40 v v ethanol 50 ultrapure water 1 Sensitiser 30 v v ethanol 4 1 w v sodium acetate 0 275 w v potassium tetrathionate and 0 5 v v glutaraldehyde i Silver stain 0 2 9o w v silver nitrate 0 062 w v HEPES 0 07 96 v v formaldehyde iv Developer 3 w v potassium carbonate 0 0012 w v sodium thiosulfate 0 025 v v formaldehyde v Stop solution 5 9o w v Tris base 2 9o v v acetic acid Method For successful silver staining use only high purity fresh chemicals Especially formaldehyde and glutaraldehyde solutions must be made fresh We never use stock solutions but make up all solut
23. Leung T Goodman B Schulenberg J Hendrickson J M Beechem R P Haugland and W F Patton 2003 Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology Proteomics 3 1128 44 Steward N R Martin J M Engasser and J L Goergen 1999 Determination of growth and lysis kinetics in plant cell suspension cultures from the measurement of esterase release Biotechnol Bioeng 66 114 21 Proteomics page 58 of 62 Medicago truncatula Handbook version March 2007 Suzuki H M Reddy M Naoumkina N Aziz G May D Huhman L Sumner J Blount P Mendes and R Dixon 2005 Methyl jasmonate and yeast elicitor induce differential transcriptional and metabolic re programming in cell suspension cultures of the model legume Medicago truncatula Planta 220 696 707 Trieu A T S H Burleigh I V Kardailsky I E Maldonado Mendoza W K Versaw L A Blaylock H Shin T J Chiou H Katagi G R Dewbre D Weigel and M J Harrison 2000 Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium Plant J 22 531 41 Unlii M Morgan ME Minden JS 1997 Difference gel electrophoresis A single gel method for detecting changes in protein extracts Electrophoresis 18 2071 2077 USDA NASS 2005 Agricultural statistics United States Government Printing Office Washington 527 pp Valot B Dieu M Recorbet G Raes M Gianinazzi S Dumas Gaudot E 2005 Identifi
24. Medicago truncatula Handbook version March 2007 Proteomics of Medicago truncatula Satish Nagaraj Zhentian Lei Bonnie Watson Lloyd Sumner Karine Gallardo Eliane Dumas Gaudot Ghislaine Recorbet Frank Robert Odile Thiery Benoit Valot Ulrike Mathesius Karsten Oelkers Related Websites Proteomics The Samuel Roberts Noble Foundation 2510 Sam Noble Parkway Ardmore OK 73402 snagaraj noble org The Samuel Roberts Noble Foundation 2510 Sam Noble Parkway Ardmore OK 73402 zlei noble org The Samuel Roberts Noble Foundation 2510 Sam Noble Parkway Ardmore OK 73402 bowatson noble org The Samuel Roberts Noble Foundation 2510 Sam Noble Parkway Ardmore OK 73402 lwsumner noble org URLEG INRA DIJON Domaine d Epoisses 21110 Breteni res France gallardo 9 epoisses inra fr UMR 1088 INRA CNRS 5184 UB Plante Microbe Environnement INRA CMSE BP 86510 21065 Dijon cedex France dumas a epoisses inra fr UMR 1088 INRA CNRS 5184 UB Plante Microbe Environnement INRA CMSE BP 86510 21065 Dijon cedex France recorbet a epoisses inra fr UMR 1088 INRA CNRS 5184 UB Plante Microbe Environnement INRA CMSE BP 86510 21065 Dijon cedex France frobert epoisses inra fr Botanical Institute University of Basel Section of Plant Physiology Hebelstrasse 1 Basel Switzerland CH 4056 odile thiery unibas ch UMR 1088 INRA CNRS 5184 UB Plante Microbe Environnement INRA CMSE BP 86510 21065 Dijon cedex France UMR d
25. N CALIBRATION VALUES Calibration is applied to selected Selected Range ample id 100 v Set AgMsstrument Default V Overwrite current file x itati i range of scans WC Quantitation Wizard g Review Results Table Calibration is applied to all scans in current sample e E 3 o o o m E Calibration is applied to all samples in the file a Current 3 568958391642671 30e 004 4 08257345105359450e 001 New 784 8282 80 7895 781 3055 782 3419 783 3188 o a d 780 0 781 0 782 0 783 0 784 0 785 0 For Help press F1 Psat e A 4 Bd A Analyst 05 or M Figure 15 Calibration of TOF MS 13 rtf WordPad 785 3338 7886 0 Calibrate spectrum Close Help 87 3427 792 8459 794 3405 794 9364 795 3480 790 7894 792 3568 793 6585 796 0 787 8468 788 3565 784 0206 789 3160 788 0 789 0 790 0 m z amu 787 0 791 0 792 0 793 0 3 793 8390 794 0 795 0 Max 243 9 counts 798 8415 797 0 788 0 9g Idle d Ready n Idle RS 4 09PM A screen shot showing the example of calibration step of TOF MS The red circles and the numbered arrows depict the steps described in 17 19 Submit the batch file by clicking the submit button It should automatically begin analyzing samples If it does not start click the start button on the tool bar to manually activate the data acquisition process During data acquisition
26. Total protein extracts were prepared from M truncatula developing seeds collected on a batch of 20 plants grown in pots at 22 19 C day night temperatures under a 16 h photoperiod at 220 wE m s light intensity with 60 70 relative humidity Individual flowers were tagged on the day of flower opening i e 1 day after pollination dap over a one month period Pods were harvested from 12 days after pollination until maturity Table 1 The developing seeds were collected on Petri dishes placed on ice to prevent any dehydration weighed Sartorius ISO 9001 Scale Quality Control Services Portland OR and rapidly frozen in liquid nitrogen To minimize the effects of heterochrony it 1s advisable to harvest grains from the central part of the pod only Between 110 and 400 mg corresponding to an equivalent of 65 seeds of fresh immature seeds were collected per stage Table 1 Seed samples were ground in liquid nitrogen using mortar and pestle The powder was homogenized and stored at 80 C until protein extraction Because M truncatula seed development is associated with important physiological changes increase in seed dry weight water loss see Table 1 total proteins were extracted from immature seeds by using 20 ul per mg of seed dry matter of a thiourea urea lysis buffer Gallardo et al 2003 Total proteins were extracted from mature seeds with 50 ul per mg of seed weight of the same buffer Le Signor et al 2005 All extractions were ca
27. a second cold mortar 4 C and homogenize with cold extraction buffer 1 10 w v of fresh root material Table 3 2 Transfer the resulting suspension to centrifuge tubes be careful to choose tubes resistant to organic solvents 3 Immediately add under hood an equal volume of biophenol pH 8 0 Tris buffered Mix thoroughly for 30 min on a shaker in a cold room 4 Centrifuge for 30 min at 12 000 g 4 C Collect the phenolic phase and add one volume of the extraction buffer Repeat the shaking step as 1n 3 and then centrifuge for 30 min at 12 000 g 4 C 5 Collect the phenolic phase and precipitate the proteins overnight at 20 C with 5 volumes of 0 1 M ammonium acetate in methanol 6 The next day pellet the proteins by swing centrifugation at 16 000 g for 30 min 4 C Rinse the pellet with methanol 3x 1ml and then acetone 3x 1 ml kept at 20 C and dry under nitrogen gas at room temperature 7 Dissolve the pellet 4 h at 20 C in O Farrell s lysis buffer Table 4 and centrifuge for 30 min at 170 000 g 20 C Supernatant can be either directly analyzed or stored at 80 C for further analyses Measure of protein content in samples This step 1s crucial for further 2D PAGE analysis since the determination of the amount of proteins effectively solubilized and applied to gels 1s required to evaluate 2D protein maps accurately and quantitatively The protein content of the supernatant is determined by Bradford s assay Bradford
28. al 3 edn CSHL Press New York santoni V P Doumas D Rouquie M Mansion T Rabilloud and M Rossignol 1999 Large scale characterization of plant plasma membrane proteins Biochimie 81 655 661 santoni V Rouquie D Doumas P Mansion M Boutry M Degand H Dupree P Packman L Sherrier J Prime T Bauw G Posada E Rouze P Dehais P Sahnoun I Barlier I Rossignol M 1998 Use of a proteome strategy for tagging proteins present at the plasma membrane Plant J 16 633 641 SAS Institute 1999 SAS STAT User s Guide SAS Institute Inc Cary NC Schaffner W and C Weissmann 1973 A rapid sensitive and specific method for the determination of protein in dilute solution Anal Biochem 56 502 514 Schulenberg B T N Goodman R Aggeler R A Capaldi W F Patton 2004 Characterization of dynamic and steady state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry Electrophoresis 25 2526 32 seigneurin Berny D Rolland N Garin J Joyard J 1999 Differential extraction of hydrophobic proteins from chloroplast envelope membranes a subcellular specific proteomic approach to identify rare intrinsic membrane proteins Plant J 19 217 228 Shorrosh B S R A Dixon and J B Ohlrogge 1994 Molecular cloning characterization and elicitation of acetyl CoA carboxylase from alfalfa Proc Natl Acad Sci U S A 91 4323 7 Steinberg T H B J Agnew K R Gee W Y
29. aldehyde solution might be old In this case re sensitize and re stain the gel in silver solution with new or a larger volume of formaldehyde Proteomics page 32 of 62 Medicago truncatula Handbook version March 2007 4 Gel analysis 4 1 Comparative software of single stained gels Nonlinear Progenesis 1 Scan gel images using a UMAX Power Look 1100 UMAX technologies or an equivalent scanner imager 1 Scanner Settings Grayscale 14 bits channel 300 dpi ii Assign a name to the image to be scanned Double click on slide icon i Preview and set the frame to be scanned iv Scan This will save image as the name provided before preview v Remember to reset name before next image or data will be lost vi If desired perform auto levels and crop images using photoshop 2 Opena new experiment in 2D Expression Progenesis Nonlinear Dynamics by selecting gels to be analyzed 3 Spot Detection 1 Detection Settings Sensitivity 9 850 Noise factor 15 Operator size 45 Background 1 ii Set to automatic splitting i Spot filter settings Area 60 Peak height 15 000 Circularity 0 2 Volume 200 000 4 Manually edit gels for artifact spots undetected spots and mis split spots 1 Use a pen size 8 i Zoom in 1 1 and manually edit ii Use high contrast for low level spots iv Use the brightness contrast slide bars to help find splits in the saturated spots 5 Select a reference gel based on the gel with the b
30. buffer E Table 8 After two successive centrifugations at 12000 and 16000 g for 20 min each the supernatant is then centrifuged at 120000 g for hour The pellet corresponding to microsomes is re solved in 600 ul of a sucrose buffer Table 9 and homogenized with a potter Membrane separation is performed on discontinuous sucrose gradient Figure 5 The protocol of Hodges et al 1972 has been modified as following 3 zh The discontinuous sucrose gradient is formed by 2 successive layers of a solution containing 3 5ml of 38 W W and 3 ml of 33 W W of sucrose in the sucrose buffer S Table 9 An aliquot 60 ul is reserved to follow the purification The remaining volume is layered on the sucrose gradient Centrifuge at 120 000 g for 2 hours in a swing rotor Membranes form rings at the 15 3396 and 33 38 interfaces and a pellet can be detected at the end of the 38 phase picture on the right in Figure 5 The 33 38 interface corresponding to a fraction enriched in plasma membrane is taken off with a syringe with a bevelled needle Sample the two other interfaces in order to follow the purification process Wash the various fractions with 4 volumes of buffer S Table 10 Centrifuge at 140 000 g for 1 hour and re suspend the fraction into 80 to 350 ul of preservation buffer P Table 11 The protein amount is determined by the method of Bradford 1976 modified by Bearden 1978 in presence of 0 0196 Triton X100 with BSA as a
31. by centrifugation at 14 000 g for 10 minutes at 10 C Occasionally Urea might crystallize if centrifugation is performed at 4 C 6 Protein concentration 1s determined by Bradford s method using the Bio Rad kit or any other method as per the lab s procedures 1 Make BSA standards at 5 ug 10 ug 15 ug 20 ug and 25 ug using a Iug ul BSA stock solution ii Add 2 uL of IEF solubilization buffer to each BSA standard to correct for urea and detergent in the protein sample ii Aliquot 2 uL of sample to three cuvettes i e the measurements should be performed in triplicate iv Mix 50 mL of Bradford s reagent 1 part of reagent to 4 parts of water v Add 1 0 mL of Bradford s reagent to each sample and standard vi Wait for 5 minutes and measure absorbance at 595 nm 7 Centrifuge the sample again to remove any residual insoluble material and dilute protein with additional resolubilization buffer as necessary to obtain a final concentration of 2 5ug ul Add bromophenol blue to a final concentration of 0 001 Proteomics page 27 of 62 Medicago truncatula Handbook version March 2007 8 Rehydrate IPG strips passively 1e no voltage applied or actively at 30V strip for at least 11 hours 9 Add wicks moistened with water and begin IEF focusing The length of time needed for IEF depends on the strip length strip pH range and sample concentration Read the instructions which come with your specific system and make sure the dy
32. cation of membrane associated proteins regulated by the arbuscular mycorrhizal symbiosis Plant Molec Biol 59 565 580 Valot B Gianinazzi S Dumas Gaudot E 2004 Sub cellular proteomic analysis of a Medicago truncatula root microsomal fraction Phytochemistry 65 1721 1732 Valot B Negroni L Zivy M Gianinazzi S Dumas Gaudot E 2006 A mass spectrometric approach to identify arbuscular mycorrhiza related proteins in root plasma membrane fractions Proteomics 6 in press van Wijk K J 2004 Plastid proteomics Plant Physiol Biochem 42 963 77 Watson B S V S Asirvatham L Wang and L W Sumner 2003 Mapping the proteome of barrel medic Medicago truncatula Plant Physiol 131 1104 23 Wessel D and U I Flugge 1984 A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids Anal Biochem 138 141 3 Wienkoop S Saalbach G 2003 Proteome analysis Novel proteins identified at the peribacteroid membrane from Lotus japonicus root nodules Plant Physiol 131 1080 1090 Wolschin F and W Weckwerth 2005 Combining metal oxide affinity chromatography MOAC and selective mass spectrometry for robust identification of in vivo protein phosphorylation sites Plant Methods 1 9 Wolschin F S Wienkoop and W Weckwerth 2005 Enrichment of phosphorylated proteins and peptides from complex mixtures using metal oxide hydroxide affinity chromatography MOAC Proteomics 5 4389
33. ceed further as for the insoluble C M fraction or in 50 ul of the extraction buffer E 19 The protein amount is determined by the method of Bradford 1976 modified by Bearden 1978 in presence of 0 01 Triton X100 with BSA as standard Add 4 volumes of cold acetone to precipitate proteins overnight They can be kept at 20 C until electrophoresis Figure 4 Schematic representation of purification and extraction of root microsomal proteins A The different fractions collected during purification of microsomes pellet Microsomal 12000 g fraction supernatant Crushed roots 16000 g pellet 100000 g Total supernatant Cytosolic fraction B Scheme of protein separation with the chloroform methanol extraction of the microsomal fraction Insoluble water methanol phase Soluble protons chloroform methanol phase proteins Proteomics page 17 of 62 Medicago truncatula Handbook version March 2007 2 7 Plasma membrane proteins Plasma membrane protein purification is performed starting from a microsomal fraction of root proteins see 2 6 10 The various membranes are separated by a discontinuous gradient of sucrose The purification process is followed by measuring several enzymatic activities selected as markers of the different types of membranes see below Valot et al 2006 1 Prepare a microsomal fraction from hundred grams of roots crush in liquid N2 and suspend in 150 ml of extraction
34. cetone mixture will usually inhibit most of the proteases It also precipitates the proteins into a pellet and removes contaminants that dissolve in the acetone e g phenolics Keeping the samples cold 1s important to improve precipitation and inhibit protein breakdown Securely close the tube and sonicate in a sonicator for example we use a Branson Ultrasonifier Place the tubes on water ice during this step because the sonication will produce heat For this step both probe sonicators which use a metal rod sticking into the sample or a cup sonicator in which the closed tube sits in a cup and sound waves are produced from the outside are OK Cup sonicators are better to prevent contamination of the samples Place the tubes on ice in the cup and sonicate 5 6 times for 10 seconds each with 30 second breaks in between Replace ice in between as it will melt The sonification 1s important to disrupt the tissue even more than what can be achieved with mortar and pestle Return tubes onto dry ice and leave for approximately 1 hr this step can be longer even over night Centrifuge the tubes either in a cooled bench centrifuge e g in the cold room at 14 000 rpm or in an ultracentrifuge at 14 000 17 000 rpm Ultracentrifugation 1s not necessary but often better if the samples are large and won t fit into an Eppendorf tube Remove the supernatant and discard Keep the pellet Add a volume of pre cooled acetone with DTT again about 10
35. d Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed Unconfirmed 3410 Unconfirmed 18 18 A M 3420 Unconfirmed 18 18 A M Master No 3403 Position 12 Master No 3403 Position 12 I Pri Spot Map Protein ID Protein AC Comment p I Pick PTM gt Confirm Mw Da J Protein of Interest Ready Focus Secondary Image INUM Figure 12 Statistical analysis of spot differences Gels are shown in the top left Mountain plots of the marked spots are shown in the bottom left A list of all proteins and the p value for their significance of expression is on the right bottom A plot of the spot abundance across all repeat gels in this case 3 1s shown on the top right the blue line is the average spot volume across the three gels In this case there were 4 different treatments shown along the x axis Proteomics page 40 of 62 Medicago truncatula Handbook version March 2007 With all experiments it is necessary to check the computer generated matches by eye and correct them if necessary All protocols for protein labeling scanning etc can be found in the Amersham Biosciences DIGE user manual Proteomics page 41 of 62 Medicago truncatula Handbook version March 2007 5 Protein identification 5 1 Tryptic digestion Lei et al 2005 Comments Wear clean gloves Work in a laminar flow hood if possible to avoid contamination Great care should be taken n
36. ds to the microsomal fraction Figure 4A 8 This pellet 1s solubilized in 3 ml of the E extraction buffer and centrifuged Ih at 100000 g Figure 4A 9 The resulting pellet is suspended in 0 1ml of E extraction buffer This microsomal fraction is then extracted according to the protocol of Ferro et al 2000 modified as following 10 Add the microsomal fraction very carefully to 0 9 ml of a mix of cold CHCl CH3OH C M 6 3 v v Mix carefully with a pipette leave on ice 30 min but shake with a vortex every 5 min 11 Centrifuge 30 min at 15000 g 4 C a very thin layer of proteins will be visible at the interface of the two phases that corresponds to the fraction of proteins insoluble in the organic mix and will be called insoluble C M fraction Figure 4B 12 Collect the two phases that contain the proteins solubilized in the organic mix solvent corresponding to the soluble C M fraction Figure 4B 13 Both fractions are dried under vacuum 14 Suspend the insoluble C M fraction in 200 ul of the 1sofocalisation buffer I Table 6 15 Lipids will be eliminated by a 30 min centrifugation at 170000 g 16 Protein amounts will be determined according to Schaffner and Weissmann s method 1973 Proteomics page 16 of 62 Medicago truncatula Handbook version March 2007 17 Samples can be kept at 20 C 18 The soluble C M fraction can be solubilized either in 30 to 50 ul of the isofocalisation buffer I Table 7 and pro
37. e G n tique V g tale IFR 87 Plate Forme de Prot omique du Moulon Gif sur Yvette France valot moulon inra fr School of Biochemistry and Molecular Biology Australian National University Canberra ACT 0200 Australia ulrike mathesius anu edu au School of Biochemistry and Molecular Biology Australian National University Canberra ACT 0200 Australia karsten oelkers anu edu au http semele anu edu au 2d 2d html http www chez com elianet http www dijon inra fr pme http www mtproteomics com http www noble org PlantBio MS index html http www pierroton inra fr genetics 2D Proteomevert page 1 of 62 Medicago truncatula Handbook version March 2007 Table of contents 1 Introduction to proteome analysis 2 Protein extraction 2 1 Cell suspension cultures 2 2 Green tissues 2 3 Developing and mature seeds 2 4 Roots and nodules 2 5 Soil grown roots inoculated or not with micro organisms 2 06 Extraction and solubilization of M truncatula root membrane proteins 2 7 Plasma membrane proteins 2 8 Combined extraction of RNA and proteins 2 9 Enrichment of phosphorylated proteins 3 Protein separations 3 1 2D gel electrophoresis 3 2 Gelstaining 4 Gel analysis 4 1 Comparative software of single stained gels Nonlinear Dynamics 4 2 Comparative software of single stained gels Image Master 4 3 Difference Gel Electrophoresis 5 Protein identification 5 1 Tryptic digestion 5 2 Mass Spectometry anal
38. e front reaches the anode In the author s lab IEF focusing 1s performed at 500 V for 1 hour 1000 V for 1 hour and 8000 V for a total of 76 500 volt hours Vhrs 1 For troublesome samples it sometimes helps to add more ampholytes up to 0 8 2 0 for Amersham strips to the resolubilization solution ll Also the strips can start to dehydrate with prolonged focusing in that case it can help to change the wicks or to pause focusing and add additional buffer to the strips 50ul for 11 cm strips and 100ul for 24cm strips 10 The focused strips can be stored at 20 C overnight or immediately equilibrated for the 2 dimension gel Overnight to over the weekend storage does not have any negative effects and freezing might even help break up the IEF gel matrix thereby increasing the efficiency of transfer of proteins to the 2 dimension gel matrix b Equilibration and second dimensional separation 12 Samples 1 Equilibrate strips in 7 M urea 375 mM Tris pH 8 8 3 SDS 10 glycerol and 50 mM DTT for 10 minutes This will aid in the reduction of cysteine bonds 2 Equilibrate strips in 7 M urea 375 mM Tris pH 8 8 3 SDS 10 glycerol and 100 mM iodoacetamide for 10 minutes This will aid in the alkylation of the reduced cystines 3 Equilibration buffer per 150 mL Urea 63 1 g 1 5M Tris pH 8 8 37 5 mL SDS 4 5 g Glycerol 15 mL DTT 0 58 g to 75 mL IAA 2 79 g to 75 mL Use 6 mL for each strip 4 Pipette marker pro
39. e tube can be left at room temperature over night but this is not necessary Once the solution looks homogenous spin for 5 min at 14 000 rpm in a bench centrifuge The proteins should go into solution Collect the supernatant into a clean Eppendorf tube Repeat this step 1 e add a little more solubilisation buffer to the pellet vortex and centrifuge again combine both supernatants Keep the pellet if you are interested in extracting some less soluble proteins from it e g membrane proteins In that case 1t 1s best to boil the pellet in a solution containing SDS and separating proteins on a 1D gel Looking at membrane proteins for subsequent 2D gel analysis 1s not recommended this 1s because the strong detergents e g SDS needed to extract membrane proteins will interfere with the isoelectric focusing See paragraph 2 6 for protocols on membrane protein extraction Measure the protein concentration of the solution We use a Bradford protein assays but others are fine as well We use a protein dilution range from 0 1 to 1 mg of bovine serum albumin BSA dissolved in the same solubilisation buffer as a standard Usually protein concentrations are between 1 5 mg ml At this stage the protein solution can be kept for several months at 80 C or can be used immediately for subsequent 1 or 2D gel electrophoresis Proteomics page 13 of 62 Medicago truncatula Handbook version March 2007 2 5 Protein extraction from soil grown roots inocula
40. ent would be assuming four biological repeats of samples and a dye swap between Cy3 and Cy5 M sample of mutant WT sample of wild type Mix mixture of all mutant all wild type samples of all repeats Cy2 Cy2 labelled etc Gel 1 MI Cy3 WTI Cy5 Mix Cy2 Gel 2 M2 Cy3 WT2 Cy5 Mix Cy2 Gel 3 M3 Cy5 WT3 Cy3 Mix Cy2 Gel 4 M4 Cy5 WT4 Cy3 Mix Cy2 After loading the samples onto 1D strips and running the first and second dimension see 3 1 the gels are then scanned at three wavelengths as shown below resulting in an overlay image Figure 8 Cy 2 Excitation at 488 nm emission at 520 nm 40 nm bandwidth Cy 3 Excitation at 532 nm emission at 580 nm 30 nm bandwidth Cy 5 Excitation at 633 nm emission at 670 nm 30 nm bandwidth Figure 8 Gel overlay of three samples in one gel imaged at three different wavelengths false colour image White spots are similarly abundant in all three samples blue red and green spots arrows are more abundant in one of the three samples The gel shown as example of M truncatula root proteins Proteomics page 38 of 62 Medicago truncatula Handbook version March 2007 The gels are cropped named with the correct extensions for analysis the spots matched Figure 10 normalized Figure 9 compared Figure 11 and statistical analysis Figure 12 done to test whether differences in spot abundance are significant over the four biological repeats EX EX no ho e en pail c
41. equivalent amount of protein can be obtained from 0 5 g of younger leaves Thirteen ml tubes are large enough for this amount of tissue For very small samples eppendorf tubes are probably large enough 3 Precipitate protein for at least 45 min at 20 C 4 Centrifuge at 14 000 x g 15 min 4 C Remove supernatant without disturbing the pellet 5 Add pre chilled 4 C 90 acetone containing 0 07 B mercaptoethanol to wash protein pellet Centrifuge at 14 000 x g 15 min 4 C Wash a total of 3 times Make sure the pellet is washed well break up protein pellet using small metal spatula with pointed tip 1f necessary The additional washes with acetone help remove residual traces of TCA that have a negative impact on isoelectric focusing 6 After the last wash leave a small amount of 90 acetone on the pellet Resuspend the pellet in the acetone and move it to an eppendorf tube Rinse the large tube with more acetone and add that to the sample in the eppendorf Spin the sample in a 4 C microfuge 14 000 x g 5 min to pellet the protein Remove the acetone 7 Air dry protein pellet in laminar flow hood The acetone must be removed from the sample but do not over dry the pellet This will cause problems lower recovery in the resolubilization step Dry until the pellet looks crisp around the edges and comes loose from the side of the tube when the tube 1s thumped The interior of the pellet should still look damp Proteomics page 6
42. es Grinding faster might cause splashing of the samples Proteomics page 4 of 62 Medicago truncatula Handbook version March 2007 2 Add a small amount of liquid Nz and swirl to break loose the ground tissue Tissue should form a lose mass 1 For extracting proteins from multiple samples place mortar in dry ice until ready for next step 11 Repeat steps 1 and 2 with the remaining samples 3 Transfer ground tissue to a 4 C mortar and add 8 mL of Tris extraction buffer i Transferring samples to a 4 C mortar will accelerate thawing and helps in homogenization ii Wait a minute or two for buffer to thaw Grind until tissue and buffer are thoroughly mixed i Prepare Tris Extraction Buffer 40 mM Tris HCl pH 9 5 50 mM MgCl 2 PVPP 125 U endonuclease mL mM PMSF iv Tris Stock 1 M at pH 9 5 stored at 4 C FW 121 4 MgCl Stock 1 M MgCb 6H O FW 203 31 PMSF Stock 100 mM in acetone made fresh FW 174 2 Dissolve 34 84 mg in 2 mL mL stock sol mL stock sol mL stock sol v Tris Extraction Buffer per 100 mL peri25 mL per150 mL Tris HCl Stock pH 9 5 4 0 mL 5 0 mL 6 0 mL MgCl Stock 5 0 mL 6 25 mL 7 5 mL 2 PVPP 2 0 g 2 5g 3 0g 125 U endonuclease mL Lot dependant PMSF Stock 1 0 mL 1 25 mL 1 5 mL 4 Pour homogenate into 13 mL polypropylene centrifuge tube vortex and sonicate 1 Vortex for 1 minute set on ice for 1 minute i1 Sonicate for 10 pulses Each Pulse 2 sec Set on ice for 3 min
43. escription is provided in 16 16 After data acquisition open the data file Zoom in the data in the region of m z 785 and then drag across the C4 isotope peak Hold down the Shift key and double click the X axis to zoom out Keep the Shift key depressed and zoom in another region of m z 785 Drag across the Cj isotope peak as shown in Figure 14 17 Right click anywhere on the spectrum and a pull down menu pops up Select Re Calibrate TOF from the menu to open the calibration window Follow the steps outlined in the Figure 15 First enter the theoretical molecular weight of peptide step 1 524 2256 for M H and 785 8384 for M H then click Calculate new calibration step 2 Calibrate spectrum step 3 and Entire file step 4 Make sure that Set as default and Overwrite current file are checked or the new calibration will not be used in the future data acquisition 18 Build a batch file in which you can enter sample names vial positions acquisition methods and the folder path where you want your data stored Save the batch file or submit it directly The current acquisition method employs a 60 minutes gradient flow 200 nl min solvent B increases from 5 to 45 in 40 minutes and then to 95 for 5 minutes and uses the IDA information dependent acquisition feature for tandem MS experiments You can edit the current method file or build your own method but should save it Proteomics page 47 of 62 Medicago truncatula Handboo
44. est focusing and most spots or based on the most representative gel within a sample 6 Spot Matching i Examine gels for high volume spots that will serve as user seeds These spots will help the matching software find real matches 1i Warp to user seeds iii Make sure selected user seeds actually helps the spot patterns match up iv Run matching v Examine matching for missed matches and mismatches 7 Manual editing of matching 1 Select high abundance spots with computer selected matched as user seeds ii If you set a user seed and it negatively impacts the spot overlay then undo the user seed i After computer matching set additional user seeds if necessary then manually match remaining spots Proteomics page 33 of 62 Medicago truncatula Handbook version March 2007 8 Background subtraction 1 Mode of non spot set to 45 9 Normalization 1 Total spot volume multiplied by 100 Proteomics page 34 of 62 Medicago truncatula Handbook version March 2007 4 2 Comparative software of single stained gels Image Master Normalization of spot volumes between seed developmental stages K Gallardo Image analysis was carried out with the ImageMaster 2D Platinum software Amersham Biosciences according to the instruction manual Protein spots were selected for quantitative analyses if they were consistently visible in three 2 D gels for at least one stage After spot matching across the different gels spot volumes we
45. gel from the glass being careful not to break the gel A helpful procedure to remove the gel from the glass plates 1s to float the gels off the glass plates in a large dish of water The gel can then be stained with Commassie fluorescent stain or silver stain See 3 2 Separation conditions for seed proteins Gel strips of 24 cm forming an immobilized non linear 3 to 10 pH gradient Immobiline DryStrip Amersham Biosciences were used for isoelectrofocusing These strips allowed a good resolution of the seed proteins that mostly range in pl from 5 to 7 with some highly basic proteins located at around pH 8 e g basic chains of legumins Twenty ul of the various protein extracts corresponding to about 300 ug proteins were added to 440 ul of rehydration buffer containing 7M Plus One urea 2M thiourea 65mM PlusOne CHAPS 20mM PlusOne DTT 0 5 pharmalyte 2 v v Triton X 100 and a trace of bromophenol blue Sigma Following strip rehydration for 7h at 20 C in the presence of the protein extract IEF was performed in the IPGphor system Amersham Biosciences for 7 h at 50 V 1 h at 300 V 2 h at 3500 V and 7 h at 8000 V Equilibrated gel strips Gorg et al 1987 were placed on top of vertical 10 w v polyacrylamide gels in a denaturing solution containing 1 w v low melting agarose 0 4 w v PlusOne SDS Amersham Biosciences 0 15M BIS Tris Sigma and 0 1M HCl For each seed sample analyzed 2D gels were made in triplicate and from two i
46. graph 2 5 Proteins can be then analyzed by 2 DE Table 12 NTES buffer for RNA extraction Initial Final per ml concentration Concentration NaCl 5M 150 mM 30 ul EDTA 0 5M 5 mM 10 ul Tris HCL pH 9 1M 50 mM 50 ul SDS 10 95 5 500 ul DEPC water 410 ul May be prepared in advance in Eppendorf tubes Add extemporaneously some mercapto ethanol 10 ul 10 ml NTES buffer Figure 6 Schematic representation of the protocol for extracting RNA and proteins simultaneously Aqueous phases A N 4 P m MRNA extraction Phenolic phases 6 Proteomics page 21 of 62 Medicago truncatula Handbook version March 2007 2 9 A batch procedure for the enrichment of phosphorylated proteins from Medicago truncatula protein extracts using Metal Oxide Hydroxide Affinity Chromatography MOAC This protocol for denaturing protein extraction and subsequent phosphoprotein enrichment can be applied to a variety of plant materials such as leaves roots nodules seeds or whole seedlings After enrichment and SDS PAGE different methods can be applied for the detection and visualization of phosphoproteins eg western blotting or fluorescent phosphoprotein specific dyes ProQ Diamond This procedure has been optimized for plant tissues and successfully applied for the identification and determination of phosphorylation sites in Arabidopsis thaliana and Chlamydomonas reinhardtii Wolschin and Weckwerth 2005 Wolschin Wienkoop et al 2005 General precauti
47. h weight FW and seed dry weight DW at specific stages of seed development is indicated Stage FW DW FW mg equivalent FW mg for 1 ml of mg seed mg seed to 1 mg DW extraction buffer 12 dap 1 71 0 21 0 21 0 02 8 14 407 14 dap 3 86 0 27 0 60 0 04 6 43 322 16 dap 4 84 0 5 0 89 0 16 5 44 272 20 dap 6 38 0 44 1 91 0 21 3 34 167 24 dap 7 22 0 08 2 80 0 06 2 58 129 36 dap 10 47 0 6 4 79 0 43 2 18 109 Dry mature 4 35 1 02 3 46 0 42 1 26 25 Proteomics page 9 of 62 Medicago truncatula Handbook version March 2007 3 5 pl gt 7 10 A PERO A l au 64 CE Hert oto oum 45 Qi tr e d o MA AL Mr ba ew kDa n 32 7m P 22 E 14 3 128 64 45 32 22 oe 14 Figure 3 Representative gels depicting the 2D resolution of M truncatula developing seeds samples of seeds harvested 12 dap A and 16 dap B are shown The protein samples were focused using 3 10 nonlinear IPG strips for the Ist dimension electrophoretically separated on a 10 acrylamide gel and stained with Coomassie Brilliant Blue G 250 Image acquisition was done using the Odyssey Infrared Imaging System Proteomics page 10 of 62 Medicago truncatula Handbook version March 2007 2 4 Protein extraction from roots and nodules Mathesius et al 2001 2003 Most parts of the root are highly vacuolated any contain contaminants that interfere with protein extraction for e
48. hizobium meliloti Bestel Corre et al 2002 and the study of Sinorhizobium meliloti and Medicago truncatula symbiosome membrane protein profile Catalano et al 2004 have also been published The effect on Medicago roots upon infection by a pathogen Aphanomyces euteiches has been analyzed Colditz et al 2004 The following sections describe the commonly utilized protocols for protein extraction from various tissues 2 dimensional gel electrophoresis 2 DE staining detection difference gel electrophoresis DIGE and the relatively new Multi dimensional protein identification technology Mud PIT followed by protein identification by mass spectrometry However this chapter describes only the protocols and if the readers want to know more about the techniques there are several reviews that discuss the technical aspects tools and hurdles of plant proteomics Hirano et al 2004 Rose et al 2004 There are also reviews that discuss the popularity Rabilloud 2002 and drawbacks Gygi et al 2000 Hamdan and Righetti 2002 Lilley et al 2002 Ong and Pandey 2001 of two dimensional gel electrophoresis 2 DE which is currently the most widely used technique in legume proteomics Proteomics page 3 of 62 Medicago truncatula Handbook version March 2007 2 Protein extraction 2 1 Cell suspension cultures method for parallel extraction of 12 Samples Lei et al 2005 Cell culture systems though considered simplified model systems
49. ion Solution 50 methanol 10 acetic acid 40 bidest water Coomassie staining Garfin 1990 version March 2007 B Phosphostain Destain Solution 20 acetonitrile 5 1M sodium acetate stock pH 4 pH adjusted with acetic acid 75 bidest water 1 rinse gel with ddH2O and stain for 45min Table 17A 2 incubate in destain solution Table 17B until background is satisfactorily reduced Table 17 A Coomassie Staining Solution 0 1 w v Coomassie Brilliant Blue G 250 50 methanol 10 acetic acid Silver staining Blum et al 1987 B Coomassie Destain Solution 10 methanol 5 acetic acid 1 rinse the gel with water and perform fixation overnight Table 18A 2 wash the gel for 30min with 30 Ethanol and three times with ddH2O for 5min three 30s rinses with ddH O ew LN a a Table 18 A Silver Fixation Solution 30 ethanol 5 acetic acid 65 ddH O C silver Staining Solution 200mg silver nitrate in 100mL ddH O Proteomics put in 50mL Sensitising Solution Table 18B for 10min stain in 50mL of Silver Staining Solution Table 18 C for 20min quickly rinse in ddH2O 10s to remove excess Staining Solution visualize spots using 50mL Developing Solution Table 18D stop with 5 acetic acid as soon as background staining becomes observable this silver stain 1s reported to be MS compatible B Silver Sensitising Solution 20mg sodium thiosulfate In 100mL bidest water saving 2mL for de
50. ion string from Fasta file Rule 20 JN E Rule to parse description string from Fasta file Rule 18 NG NI x Rule to parse accession string from local reference file Rule 243 ALA AE ES x Source and parse rule for full text report optional Host localhost Port fso Path d INETPUB MASCOT x cgi lms getseq exe MSDB ACCES Rule 9 X XC A Z 1 9 O Test this definition Delete this definition Name NCBlnr Active Inactive C Path d INETPUB MASCOT sequence NCBlnr current NCBInr fe AA C NAC m Mem map MV Memlock M Bil ie eee er zl Es Local intranet Start SIA DY B Exploring msi Mascot Protein Identificati Mascot Database M amp seting up MtGI051903 3 Desktop gt ADDY 11 21 4M Figure 16 Mascot database maintenance page Proteomics page 49 of 62 Medicago truncatula Handbook version March 2007 2 Click New Database button if you need to create one Type in the name of the new database path select AA if it is a protein database or NA if it is a nucleotide database Choose appropriate rules for different type of database For FASTA databases use rule 6 or other appropriate rule to parse the accession number and rule 7 or other appropriate rule to the description from the FASTA file 3 Click Test This Definition button at the bottom of the page Mascot will then compress the first 5 and the final 5 sequences If no problem meaning no
51. ions just before use It 1s easier to stain in photographic trays on an orbital shaker 50 rotations per minute at room temperature under a fume hood 1 Directly after SDS PAGE fix gels three times for 30 min each in fixative solution 1i Transfer to sensitiser for 16 h avoid evaporation i Wash the gel at least six times in ultrapure water for 30 min each Be sure to wash several times to remove glutaraldehyde iv Incubate gels with silver staining solution for 2 h in the dark To wash off the silver quickly rinse for 10 s in ultrapure water If this washing step 1s done for any longer the silver will disassociate from the protein spots v Develop gels for 5 to 7 min and stop the development by replacing the developer with stop solution The development should be stopped when no more new spots become visible and before the background of the gel becomes dark Timing is important because gels of different runs need to be comparable in staining vi Leave the gels in stop solution for no longer than 20 min to avoid colour changes in the silver stain Proteomics page 31 of 62 Medicago truncatula Handbook version March 2007 vii Wash gels in distilled water several times scan and store sealed in plastic pouches in a few mL of 1 methanol to help prevent microbial contamination Gels can be stored at room temperature or at 4 C for many years Note When silver staining results in only faintly stained spots the form
52. k version March 2007 as a different name Do not overwrite the existing method A good starting point for building your own method 1s electrospray voltage 2400 v mass scan range m z 100 1500 charge state for parent ion selection 2 to 5 intensity threshold for tandem MS 10 counts s precursor ions exclusion 90s using a window of 6 amu to minimize the redundancy in tandem mass spectra following IDA data acquisition Analyst QS TOF MS Experiment 1 10 423 to 11 035 min from calibration wiff 18 x B File Edit View Tools Explore Window Script Help l x le 2 Wg ir Lyg Jexplore Mode a amp zhentian Lei a X lm Ir EE JAS Se PK ERRK MwA eM hei HBL RELENING F xd xi TOF MS Experiment 1 10 423 to 11 035 min from calibration wiff aH Configure a 3 56895839 164267 130e 004 1024 08257345105359450 e 001 R Security Configuration gH Hardware Configuration Report Template Editor uh Tune 1 f Resolution Optimization EN Quantitative Optimization ce Manual Tuning Mh TOF Mass Calibration 3 Acquire E Build Acquisition Method Build Acquisition Batch TOF Calibration Reference T able Z7 Express View Explore 1 785 8387 v tolerance fi m 4 Calculate new calibrations m snms S Restore original calibration f Open Data File Open Compound Database L Quantitate Te Pee EwemalCalbraion M SAVE CURRENT CALIBRATIO
53. l approximately 1 2 of the available lysine residues about 1 residue per protein All three dyes have approximately the same size so that they add the same mass to each protein 500 Da They do not change the charge of the proteins do not interfere with 2DE or with mass spectrometry For spot picking it 1s best to counter stain the gels with Coomassie Brilliant Blue The workflow for DIGE 1s as follows e Extract proteins as usual but elute into a different buffer see below e Split samples into different tubes sample to be labeled with C3 or C5 and a tube containing the mixture of all samples Label proteins Run straight away on 1D and 2D Scan gels on a laser scanner immediately after second dimension 1s finished Generate gel analysis files and analyse using software Counter stain gels with coomassie and keep for later mass spectrometry Proteomics page 36 of 62 Medicago truncatula Handbook version March 2007 Pooled internal e ON standard 9 9 Label with Cy2 Protein extract 1 a 9 2 DE Label with Cy3 7 Protein extract 2 29 Label with Cy5 9 9 Label proteins and mix Scan at 3 Differential samples before 1D wavelengths display Figure 7 Principle of DIGE Three samples are labeled with Cy Dyes before separation by 1D and 2D electrophoresis Adapted from Amersham Biosciences Ettan DIGE User Manual Protein extraction of proteins for DIGE labelling Protein extraction should proceed as normal until the
54. ments This solution will be very concentrated Only add the water at the end because there are a lot of dry ingredients They will dissolve better if you hold the tube into a sonic bath for a few minutes with shaking or place on a rotating wheel for a few minutes If making a stock solution and freezing at 80 C do not add the protease inhibitors to the frozen stock add fresh on the day of use to defrosted solution We have found these protease inhibitors quite sufficient although other ones could be used Generally it doesn t make a difference to add them or leave them out because the TCA will inhibit most proteases Proteomics page 11 of 62 Medicago truncatula Handbook version March 2007 10 11 12 13 14 Have ready a bucket of dry ice and a bucket of water ice liquid nitrogen cleaned and pre cooled mortar and pestle spatula and centrifuge tubes Grind tissue optional with a bit of fine glass powder in liquid nitrogen in a clean mortar and pestle Thorough grinding is a very important step Usually we grind tissue for at least 5 minutes until a fine white powder appears scrape the ground tissue out of the mortar with a pre cooled spatula into a pre cooled best on dry 1ce Eppendorf tube or ultracentrifuge tube needs to be acetone resistant for example Beckman Polyallomer tubes Add approximately 10 times volume of the pre cooled TCA acetone solution to the ground tissue Vortex Keep on dry ice The TCA a
55. n by adding 25 ul of 10 formic acid note pH change Allow to stand for 15 minutes 14 Recover supernatant one can sample this solution directly for peptide mass mapping or continue extracting additional peptides We suggest further extraction 15 Extract gel plug band twice with 25ul of 5096 5096 acetonitrile 50mM ammonium bicarbonate for 15 minutes to recover additional peptides Pool with supernatant from step 14 16 Extract with 25 ul to 50 ul of 100 acetonitrile Pool with supernatant from step 14 17 Concentrate to 5 ul or dryness in vacuum centrifuge Concentration to dryness is more convenient but may result in some loss of peptides which will not resolubilize following precipitation due to drying 18 Dried peptides can be stored at 20 C or 80 C for long periods of time lyr 19 Immediately prior to mass analysis dissolve dried peptides in 50 50 acetonitrile 19o formic acid Alternatively 0 1 trifluoroacetic acid can be used but TFA can reduce mass spectral ionization efficiency through ion pair formation Proteomics page 43 of 62 Medicago truncatula Handbook version March 2007 5 2 Mass Spectral analysis via nano LC QTOF MS MS Over the past decades the rapid development of mass spectrometry MS has made it an indispensable tool in biological research The introduction of soft ionization techniques such as electrospray ionization ESI and matrix assisted laser desorption ionization MALDI has made it
56. nd R Scalla 1986 Inhibition of adenosine triphosphatase activity from a plasma membrane fraction of Acer pseudoplatanus cells by 2 2 2 trichloroethyl 3 4 dichlorocarbanilate Plant Physiol 80 782 785 Blum H H Beier and H J Gross 1987 Improved silver staining of plant proteins RNA and DNA in polyacrylamide gels Electrophoresis 8 93 99 Bomhoff G H and M Spencer 1977 Optimum pH and ionic strength for the assay of the cytochrome c oxidase from pea cotyledon mitochondria Can J Biochem 55 1114 1117 Borner G H H K S Lilley T J Stevens and P Dupree 2003 Identification of glycosylphosphatidylinositol anchored proteins in Arabidopsis A proteomic and genomic analysis Plant Physiol 132 568 577 Bradford M M 1976 A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding Anal Biochem 72 248 254 Proteomics page 55 of 62 Medicago truncatula Handbook version March 2007 Canovas F M E Dumas Gaudot G Recorbet J Jorrin H P Mock and M Rossignol 2004 Plant proteome analysis Proteomics 4 285 298 Catalano C M W S Lane and D J Sherrier 2004 Biochemical characterization of symbiosome membrane proteins from Medicago truncatula root nodules Electrophoresis 25 519 31 Chen G T G Gharib C C Huang J M Taylor D E Misek S L Kardia T J Giordano M D Iannettoni M B Orringer S M Hanash and D G Beer 2002 Disc
57. ndependent protein extractions Gels were stained with Coomassie Brilliant Blue G 250 Bio Rad Hercules CA USA according to Mathesius et al 2001 Image acquisition was done using the Odyssey Infrared Imaging System LI COR Biosciences GmbH Germany at 700 nm with a resolution of 169 um This scanning system allows a highly sensitive detection of proteins stained with Coomassie Blue in 2D gels Proteomics page 29 of 62 Medicago truncatula Handbook version March 2007 3 2 Gel staining a Coomassie staining adapted from Anderson et al 1991 1 Stain gels with Coomassie Brilliant Blue G 250 CBB G 250 1 ii iil IV Vl Vll viii IX X Xl Fix gels for 4 hours in 8 L of 40 methanol and 2 phosphoric acid The quantity mentioned here is for 12 gels and is for using with an automated DoDeca stainer large BioRad Fixing Solution per 8 0 L Methanol 3200 mL Phosphoric acid 160 0 mL Add MilliQ water to make up the volume to 8 0 L Equilibrate gels for 12 hours in 8 L of 34 Methanol 12 ammonium sulphate and 2 phosphoric acid Equilibrating Solution per 8 0 L Methanol 2720 mL Phosphoric acid 160 mL Ammonium sulphate 960 g Add milliQ water to a final volume to 8 0 L stain the gels with 8 L of CBB G 250 for 3 4 days The final concentration of G 250 is I g L or 0 1 in solution of 34 Methanol 12 ammonium sulphate and 2 phosphoric acid solution 2 Shorter staining times are
58. ning to monitor the success of the enrichment Firstly Phosphoproteins are detected with the Pro Q Diamond Phosphoprotein gel stain Steinberg Agnew et al 2003 Schulenberg Goodman et al 2004 Coomassie staining and if necessary silver staining are accomplished afterwards If not otherwise stated 80mL of staining and washing solutions were used Alternatively Western Blotting can be performed Detection of phosphoproteins with the Pro Q Diamond Gel Stain Molecular Probes Eugene 1 remove excess running buffer after SDS PAGE by washing 15min with ddH O incubate for 15min in fixation solution Table 16 A renew solution and perform fixation overnight This step 1s considered to be important for the specificity of the staining remove fixation solution by three 10 min incubations in ddH2O stain for 2h in 50mL Pro Q Diamond gel stain protect the trough during staining and destaining from light destain for 3h overall renew the destain solution every hour Table 16 B substitute the destain solution with ddH2O before imaging 0 There are different ways of imaging the Pro Q stain If there is no appropriate fluorescent scanner available the smaller UV excitation maximum at 380nm can be used in standard UV Geldocs You will need an additional broad range amber filter Also you can use the Dark Reader Clare Chemicals Dolores 2 9 9 URUM Proteomics page 25 of 62 Medicago truncatula Handbook Table 16 A Phosphostain Fixat
59. o development comparable to that observed in vivo Plant Science 170 1052 1058 Gallardo K C Le Signor J Vandekerckhove R D Thompson and J Burstin 2003 Proteomics of Medicago truncatula seed development establishes the time frame of diverse metabolic processes related to reserve accumulation Plant Physiol 133 664 82 Gana J A N E Kalengamaliro S M Cunningham and J J Volenec 1998 Expression of beta amylase from alfalfa taproots Plant Physiol 118 1495 506 Garfin D E 1990 One dimensional gel electrophoresis Methods Enzymol 182 425 4 G rg A W Postel J Weser S G nther J Strahler S Hanash and L Somerlot 1987 Elimination of point streaking on silver stained two dimensional gels by addition of iodoacetamide to the equilibration buffer Electrophoresis 8 122 124 Guo L R A Dixon and N L Paiva 1994 Conversion of vestitone to medicarpin in alfalfa Medicago sativa L 1s catalyzed by two independent enzymes Identification purification and characterization of vestitone reductase and 7 2 dihydroxy 4 methoxyisoflavanol dehydratase J Biol Chem 269 22372 22378 Proteomics page 56 of 62 Medicago truncatula Handbook version March 2007 Gygi S P G L Corthals Y Zhang Y Rochon and R Aebersold 2000 Evaluation of two dimensional gel electrophoresis based proteome analysis technology Proc Natl Acad Sci U S A 97 9390 5 Gygi S P Y Rochon B R Franza and R Aebersold 1
60. of 62 Medicago truncatula Handbook version March 2007 Figure 2 Representative gels depicting the 2D resolution of M truncatula leaf and stem Samples of leaf tissue A and stem tissue B were harvested and processed for 2 DE separation The protein samples were focused using 3 10 linear IPG strips for the 1 dimension electrophoretically separated on a 12 acrylamide gel and stained with Coomassie Proteomics page 7 of 62 Medicago truncatula Handbook version March 2007 8 The protein pellet can be stored in the freezer at this step 9 Resolubilize the protein pellet in an appropriate volume of solubilization buffer See 3 1 A rule of thumb for determining the amount of solubilization buffer needed is to add 0 5 1 ml buffer g starting material but this depends on the protein concentration of your starting material older tissue usually has a lower protein concentration per gram dry weight and whether you plan to silver or Coomassie stain the gel 10 Pellet insoluble material 14 000 x g for 10 min 10 C The urea may precipitate if you spin at 4 C 11 Transfer supernatant to new test tube and check protein concentration using the Bradford assay See 3 1 a 6 12 Proteins can then be resolved by 1D or 2D gel electrophoresis as described below See 3 1 Proteomics page 8 of 62 Medicago truncatula Handbook version March 2007 2 3 Total protein extraction from developing and mature seeds Gallardo et al 2003
61. ological material we have developed a protocol in which proteins can be analyzed from the same root sample as a mRNA population in order to make simultaneous proteome and transcriptome profiling possible This protocol was validated to analyze the early stages of the AM symbiosis Dumas Gaudot et al 2004 Figure 6 First day 1 Take Ig of roots from plantlets freshly harvested or stored at 80 C 2 Crush the roots in a cold mortar with liquid N gt into a very thin white powder 3 Pour this material in a N cooled tube wash the mortar with little liquid N2 to collect all the root material and leave the liquid Nz to evaporate without letting the powder dry 4 Add2 ml of NTES buffer Table 12 per g of roots shake with a vortex until obtaining a very viscous liquid Add 1 volume of phenol chloroform isoamylic alcohol 25 24 1 and shake again with a vortex until obtaining a whitish liquid Sample 4 X 1000 ul in 4 Eppendorf tubes 7 Centrifuge at 12000 g during 15 min at 20 C 8 Collect the supernatant 400 to 500 ul Avoid taking the intermediary phase and keep the phenolic phase on 1ce for the protein extraction 9 Repeat the steps 6 to 9 twice 10 Keep the supernatant about 200 ul to proceed to a chloroform extraction of RNA and put aside the phenolic phases 2 and 3 at 20 C 11 Centrifuge 12000 g for 10 to 15 min at 20 C 12 Collect the aqueous phase and add 0 05 v acetic acid 1 M et 0 7 cold ethanol 96 kept at 20
62. ommunicate with it 9 Restart the computer to clear any programs that may run in the background and free up the memory 10 Launch the Analyst software 11 Activate the hardware profile LC MS Three icons MS Famos and Ultimate will show up in yellow at the low right corner 12 Equilibrate the LC system with the method that you will use later to acquire data The equilibration washes the injection syringe and begins to pump solvents through the column to equilibrate it 13 Click tune in the left column the navigator and then manual tuning Open the manual tuning file by clicking the file opening 1con and select method Manual tuning dam 14 Begin the manual tuning by clicking the start button The purpose of manual tuning 1s to obtain an optimal baseline Adjust the electrospray needle position to optimize the intensity of baseline Under the current flow rate and mobile phase compositions the baseline should be between 5 000 to 20 000 cps Do not spray directly onto the orifice This will not only reduce the signal intensity but also clog the orifice more quickly over time 15 After manual tuning exit the tune mode by clicking the T 1con on the tool bar Calibrate the TOF with 250 fmol Glu Fibrinopeptide B 50 fm uL SuL pickup Sigma cat F 3261 Acquire both TOF MS and tandem MS data using the existing acquisition method glufib calibration dam Proteomics page 46 of 62 Medicago truncatula Handbook version March
63. ons e prepare protein samples freshly do not freeze proteins or store in aqueous solution e incase storage is inescapable store as dried pellets overnight e worked with chilled solutions 4 C use protease and protein phosphatase inhibitors e be careful with inhibitors as they are very toxic e work under the fume hood for the denaturing extraction procedure Protease inhibitors and protein phosphatase inhibitors e Plant Protease Inhibitor Cocktail Sigma Taufkirchen contains AEBSF 1 10 Phenanthroline Pepstatin A Leupeptin Bestatin and E 64 e Benzamidine and EDTA both 1mM can be additionally used as protease inhibitors as not included in the cocktail e Protein Phosphatase inhibitors such as Sodium fluoride 60mM sodium orthovanadate 1mM and Mikrocystin 0 3uM are known to be compatible with the enrichment Preparation of biological material 1 freeze plant material in liquid nitrogen 2 grind to a fine powder you can add a spatula tip of quartz sand 3 store at 80 C until usage 4 weigh 1g of frozen powder into 15ml Falcon tube 3 Denaturing Protein Extraction Extraction 1 add 7 5ml chilled Phenol TE buffer saturated Phenol to the frozen plant material 2 add 2 5ml chilled Denaturing Extraction Buffer DEB Table 13 3 vortex mixture thoroughly and make sure no frozen clots are left keep chilled 4 incubate on a rotator at 4 C cold room for 30min 5 centrifuge at 4 C and 3 250g for 10min pre cool the centrif
64. optional however longer staining times provide enhanced sensitivity and dynamic range staining Solution per 8 0 L CBB G 250 8 0 g Methanol 2720 mL Phosphoric acid 160 mL Ammonium sulphate 960 g Dissolve 8 g of CBB G 250 in 2000 mL of methanol stock 4 mg mL Stir overnight To this add the remaining volume 720 ml of methanol phosphoric acid ammonium sulphate and make up the volume to 8 0 L using milliQ water Drain CBB G 250 from the staining containers and replace with 10 methanol Shake for 1 hour to remove residual CCB G 250 on the walls of the container Drain and add 10 L of H20 plus 2 Kimwipes Change Kimwipes and allow destaining overnight Kim wipes are added to absorb the Commassie and speed up the destaining process This addition may be a source of contamination during mass spectrometry analysis The following morning add 8 0 L of 5 acetic acid and equilibrate for 1 hour The gels can be stored at 4 C after scanning see 4 Gel Analysis Alternative method Neuhoff et al 1985 Electrophoresis 6 427 448 b Silver staining Proteomics Silver staining is a sensitive staining method but the disadvantages of its use are that after silver staining gels cannot be blotted proteins cannot be identified subsequently by mass spectrometry and the silver staining 1s not very quantitative Silver 10ns complex to Glu Asp and Cys residues in proteins Formaldehyde under alkaline conditions reduces the Ag to
65. or 3 250g for 10 min at RT Proteomics page 24 of 62 Medicago truncatula Handbook version March 2007 5 carefully remove upper phase with a Pasteur pipette do not touch the interphase 6 add 3 volumes of methanol and mix until homogeneity of turbidity 7 spin for 3 250g for 10 min at RT 8 carefully remove solution 9 dry pellet at room temperature for 20min 10 resuspend every pellet in 40L singly concentrated SDS sample buffer 11 heat the glass test tube 1n the burner flame for a few seconds 12 spin the solution down and load the proteins on a gel 13 Adjust your loadings onto the gel so that you have similar amounts of total protein from every sample Positive and negative controls for the detection of phosphoproteins Common molecular weight markers can be used as positive and negative controls for the staining procedure The commercially available molecular weight markers often contain ovalbumin which is phosphorylated at four serine residues Swiss Prot entry P01012 Standards for threonine or tyrosine phosphororylated proteins are also available eg EGF stimulated cell lysate from Upstate For an immuno staining it 1s very handy to use the appropriate phosphoamino acid as antibody inhibitors Pre incubate the inhibitor and antibody before giving it to the membrane Cross reactivity and unspecificity especially of secondary antibodies can be easily detected in this manner Protein detection Perform multiplexed stai
66. ordant protein and mRNA expression in lung adenocarcinomas Mol Cell Proteomics 1 304 13 Colditz F O Nyamsuren K Niehaus H Eubel H P Braun and F Krajinski 2004 Proteomic approach identification of Medicago truncatula proteins induced in roots after infection with the pathogenic oomycete Aphanomyces euteiches Plant Mol Biol 55 109 20 Cook D R 1999 Medicago truncatula a model in the making Curr Opin Plant Biol 2 301 304 Daniell T and R Edwards 1995 Changes in protein methylation associated with the elicitation response in cell cultures of alfalfa Medicago sativa L FEBS Lett 360 57 61 Dumas Gaudot E Amiour N Weidmann S Bestel Correl G Valot B Lenogue S Gianinazzi Pearson V and Gianinazz S 2004 A technical trick for studying proteomics in parallel to transcriptomics in symbiotic root fungus interactions Proteomics 4 451 453 Edwards R and R A Dixon 1991 Purification and characterization of S adenosyl L methionine caffeic acid 3 O methyltransferase from suspension cultures of alfalfa Medicago sativa L Arch Biochem Biophys 287 372 9 Fodor I K D O Nelson M Alegria Hartman et al 2005 Statistical challenges in the analysis of two dimensional difference gel electrophoresis experiments using DeCyder Bioinformatics 21 3733 3740 Gallardo K C Kurt R Thompson and S Ochatt 2006 In vitro culture of immature M truncatula grains under conditions permitting embry
67. ot to touch gels allow hairs to fall on gels or allow wools to come into contact with gels prior to staining or digestion All these can contribute protein contaminants keratins which will appear and interfere with the final protein identification 1 Excise gel bands or spots Manual excision of spots can be achieved using a pipette style spot picker The Gel Company San Francisco CA and automated excision can be performed using commercial equipment Investigator Propic robotic workstation Genomics Solutions Ann Arbor MI 2 It is highly recommended that a positive and negative control be conducted in parallel to all analyses The positive control can be a marker or known protein while the negative control should be a spot band from a blank region of the gel 3 Wash twice with 50 ul of 18 Mohm water for approx 15 minutes 4 Wash sufficient times with 50 ul of a 5096 5096 solution of acetonitrile 50mM ammonium bicarbonate to remove all stain from gel plug Each wash should be approximately 30 minutes in length Two washes should be sufficient for moderately intensity bands 5 Wash and dehydrate with 50 ul spot to 100 ul band of acetonitrile until gel plugs turn opaque and dramatically shrinks in size generally 20 minutes 6 Remove all liquid from opaque gel plugs 7 Prepare trypsin solution using sequence grade modified bovine or porcine Trypsin Roche Promega Prepare stock trypsin solution by dissolving 20 to 25 mg
68. oteins that are resolvable and possible to identify Altogether several thousand proteins have been identified from M truncatula and this number will rise Any user of proteomics will have to keep the advantages e g ability to visualize protein isoforms post translational modifications and ability to quantify the real gene expression and disadvantages technical limitations 1n mind Acknowledgements Benoit Valot was financially supported by a MNERT Minist re de l Education Nationale et de la Recherche Technique grant This work was funded through the AIP INRA CNRS Medicago truncatula a model for plant symbioses 1997 99 and the Conseil R gional de Bourgogne 04 516 CP09 5327 We are grateful to Marc Dieu C line Henry Luc Negroni Michel Rossignol Nicolas Sommerer and Michel Zivy who kindly performed mass spectrometry analyses Satish Nagaraj Zhentian Lei Bonnie Watson and Lloyd Sumner gratefully acknowledge financial support from The National Science Foundation Plant Genome Research Program DBI 0109732 and The Samuel Roberts Noble Foundation Proteomics page 51 of 62 Medicago truncatula Handbook version March 2007 Ulrike Mathesius and Karsten Oelkers acknowledge funding from the Australian Research Council of Excellence for Integrative Legume Research CE0348212 and for a Research Fellowship for UM from the Australian Research Council DP0557692 Proteomics page 52 of 62 Medicago truncatula Handbook
69. otosynthetic proteins also dominate the gels and prevent the visualization of lower abundant proteins due to the limited dynamic range of common visualization stains In addition plant cells contain many proteases One extraction method used to overcome some of these problems is precipitating with trichloroacetic acid TCA in acetone Homogenizing the sample in 10 TCA dissolved in acetone almost immediately inactivates proteases and precipitates proteins while providing a means of delipidating membranes and releasing membrane associated proteins This procedure also allows interfering substances to be washed from the precipitated proteins and provides a clean sample for isoelectric focusing Please note that a strong resolubilizing buffer must be used to ensure efficient resolubilization of the precipitated protein Be careful not to overdry the sample before resolubilization Complete drying removes residual water trapped within the protein pellet and makes resolubilization considerably more challenging Representative 2 DE gels of leaf and stem tissues are shown in Figure 2 A and B 1 Grind frozen tissue very finely in liquid nitrogen with a mortar and a pestle pre chilled with liquid nitrogen 2 Transfer to a polypropylene tube containing chilled 4 C 10 TCA in acetone plus 0 07 D mercaptoethanol and vortex The size of the tube will be determined by your sample For older leaves and Coomassie staining use 2 g of tissue and 25 ml tubes An
70. ourea 2M Triton X 100 0 5 v v ASBIA 2 w v TBP 2 mM Proteomics Tris MES pH 8 Tris MES pH 7 2 Tris MES pH 7 3 Tris MES pH 6 5 Table 8 Extraction buffer E 50 mM 0 5 M 20 mM 0 1 mM ImM Table 9 Sucrose buffer S 1 mM 1 mM 15 9o w w Table 10 Sucrose buffer S2 10 mM 250 mM 1 mM mM 1 mM 1 mM Table 11 Preservation buffer 10 mM 250 mM 2mM page 19 of 62 Medicago truncatula Handbook version March 2007 2 8 Combined extraction of RNA and proteins Functional genomics i e the systematic elucidation of coding sequences in the genome has mainly been investigated at transcript or protein levels separately However in most cases there is little correspondence between these two components Chen et al 2002 Gygi et al 1999 This is mainly due to post translational modifications that affect protein behaviour and the fact that both levels are measured independently in separate sample extracts Few biological systems are sufficiently synchronised or amenable to direct comparative transcriptome proteome analyses and they are mainly limited to isolated cell cultures Chen et al 2002 In the case of complex situations involving two or more organisms synchronisation of developmental processes is often difficult to establish The root fungus symbiosis represented by arbuscular mycorrhiza associations 1s one example of such a case To circumvent the problems inherent to a limited amount of bi
71. pectives of proteomics in dicot plants Part III Unraveling the proteomes influenced by the environment and at the levels of function and genetic relationships J Chromatogr B Analyt Technol Biomed Life Sci 815 137 45 Amersham Biosciences Ettan DIGE User Manual http www amershambiosciences com Anderson N L R Esquer Blasco J P Hofmann and N G Anderson 1991 A two dimensional gel database of rat liver proteins useful in gene regulation and drug effects studies Electrophoresis 12 907 30 Baier R K Schiene B Kohring E Flaschel and K Niehaus 1999 Alfalfa and tobacco cells react differently to chitin oligosaccharides and sinorhizobium meliloti nodulation factors Planta 210 157 64 Bearden JC 1978 Quantitation of submicrogram quantities of protein by an improved protein dye binding assay Biochim Biophys Acta 533 525 529 Bell C J R A Dixon A D Farmer R Flores J Inman R A Gonzales M J Harrison N L Paiva A D Scott J W Weller and G D May 2001 The Medicago Genome Initiative a model legume database Nucleic Acids Res 29 114 7 Bestel Corre G E Dumas Gaudot V Poinsot M Dieu J F Dierick T D van J Remacle V Gianinazzi Pearson and S Gianinazzi 2002 Proteome analysis and identification of symbiosis related proteins from Medicago truncatula Gaertn by two dimensional electrophoresis and mass spectrometry Electrophoresis 23 122 37 Blein J X de Cherade M Bergon J Calmon a
72. protein levels Gygi et al 1999 The lack of correlation 1s not surprising considering the important roles of post translational modifications controlled proteolysis protein sorting and protein protein interactions on the regulation of active enzyme levels Thus proteomics has become a critical complement to mRNA data and a better systems biology view of plant and legume biology Several plant proteomics reviews have been published Canovas et al 2004 Rossignol 2001 including sub cellular proteomics van Wijk 2004 A more recent three part review discusses proteomics of dicot plants in great detail Agrawal et al 2005a Agrawal et al 2005b Agrawal et al 2005c A large number of groups have published protein reference maps for specific Medicago tissues that include roots stems flowers seed pods and cell suspension cultures Lei et al 2005 Mathesius et al 2001 Watson et al 2003 Additional organ specific proteome analyses have been reported for specific stages of seed filling and seed development Gallardo et al 2003 and for somatic embryogenic tissue culture cells Imin et al 2004 A major factor that accentuates Medicago truncatula as a model plant 1s its ability to associate with nitrogen fixing rhizobium bacteria and mycorrhizal fungi Unlike in Arabidopsis this enables the study of plant symbioses in Medicago Proteomics of roots inoculated with mycorrhizal fungi Glomus mosseae or nitrogen fixing bacteria Sinor
73. re solubilise each pellet in 0 8ml Phosphoprotein Loading Buffer Table 14 by vortexing 9 Since it 1s a denaturing extraction make sure you vortex very thoroughly to renature the proteins 10 you might need to clarify the solution by transferring the supernatant to a 2mL tube and spin insoluble compounds down 13 350g 4 C 2min 11 determine the protein concentration with the Bradford assay 12 one gram of leaf powder yields approximately 0 5mg of protein So ee a Metal oxide hydroxide affinity chromatography MOAC Preparation of the original protein sample 1 use Img protein in 1 5ml PLB per enrichment preparation 80mg aluminium hydroxide 2 save around 100ul of this original protein sample to monitor the success of your enrichment 3 continue to work chilled However you will find that it 1s difficult to keep the PLB at 4 C since urea is precipitating Table 14 Phosphoprotein Loading Buffer PLB 8M Urea 30mM MES Proteomics page 23 of 62 Medicago truncatula Handbook version March 2007 200mM Sodium glutamate 200mM Potassium aspartate 20mM Imidazole 0 25 CHAPS Inhibitors see above Adjustment of the pH with HCI to 6 1 Do not use phosphoric acid to adjust the pH Equilibration of the matrix 1 weigh 80mg aluminium hydroxide Sigma Taufkirchen in a 2ml cup 2 add 1 5ml PEB mix on vortex 3 centrifuge at 13 350g and 4 C for 2min 4 remove the supernatant Binding phosphoproteins to the matrix
74. re collected and manually normalized to profile individual spot quantity during seed development Because this process is associated with many changes at the protein level e g storage proteins which account for up to 70 of the total nitrogen in mature legume seeds accumulate in abundance the probably most reliable scaling procedure in each 2D gel is a normalization of the volume i e abundance of each spot to the volume of a set of housekeeping proteins consistently accumulated during seed development Such internal reference proteins were selected by comparing qualitatively silver and Coomassie blue stained 2 D gels across the developmental stages Gallardo et al 2003 Ten of them were identified by mass spectrometry as corresponding to calctum ATPase RuBisCO subunit binding proteins Heat shock proteins glucose 6 phosphate isomerase putative aminoaldehyde dehydrogenase and putative TPR repeat protein TC91689 Table 19 shows the experimental characteristics of these reference proteins in 2D gels from total protein extracts of developing seeds Interestingly most of them are known to play housekeeping roles in the cell such as maintenance of pH Ca2 homeostasis protein folding or glycolysis suggesting a function for these proteins throughout seed development Following spot volume normalization differences in the abundance of each spot among the different samples was analyzed by ANOVA one way analysis of variance and a Student Ne
75. rried out at 4 C in 1 ml of the thiourea urea lysis buffer Table 1 containing 7M PlusOne urea Amersham Biosciences Orsay France 2M thiourea Sigma St Quentin Fallavier France 62 mM PlusOne CHAPS 3 3 cholamidopropyl dimethylammonio 1 propane sulphonate Amersham Biosciences 1 v v carrier ampholytes pharmalyte pH 3 to 10 Amersham Biosciences 0 24 v v Triton X 100 Sigma This extraction buffer also contained nucleases 60 Units DNase I from Roche and 5 8 Kunits RNase A from Sigma 21 mM Tris HCl 16 5 mM Trizma base Sigma and the protease inhibitor cocktail complete Mini from Roche Diagnostics GmbH Mannheim Germany After stirring the samples for 30 min at 4 C 8 mM dithiothreitol PlusOne DTT Amersham Biosciences was added The protein extracts were stirred for 20 min at 4 C then centrifuged 20 000g 10 min 4 C The supernatant was submitted to a second clarifying centrifugation as above The final Supernatant corresponded to the total protein extract Protein concentration was measured according to Bradford 1976 by using the Bio Rad Marnes la Coquette France Protein Assay compatible with the reducing and denaturing agents used to prepare all protein extracts Bovine serum albumin was used as a standard A representative gel of developing seed proteins 1s shown in Figure 3 Table 1 Seed fresh weight used to extract total proteins in 1 ml of the thiourea urea lysis buffer Correspondence between seed fres
76. standard Fractions should be kept at 80 C before analysis Figure 5 The different steps of plasma membrane purification by sucrose gradient The fraction containing the plasma membrane is sampled at the 33 38 interface On the right a picture showing a separation after centrifugation Valot unpublished result 33 38 33 2h fraction 38 100000 g Microsomal Sucrose hb A fraction gradient Proteomics page 18 of 62 Medicago truncatula Handbook Enzymatic assays version March 2007 Marker enzymes for plant cell membranes must be assayed in the 3 fractions obtained after sucrose gradient partitioning The K Mg ATPase activity sensitive to SW26 was assayed as a marker for the PM whereas the insensitive one was used to follow the other membranes Blein et al 1986 The pyrophosphatase inosine diphosphatase NADH cytochrome c reductase insensitive to antimycin A and cytochrome c oxidase were used as markers for the tonoplast the Golgi apparatus the endoplasmic reticulum and mitochondria respectively Joyce et al 1988 Bomhoff G H and Spencer 1977 Assays should be performed on 3 independent sucrose partitioning experiments in a sufficient number of replicates Table 5 Extraction buffer E Tris HCl pH 7 5 0 5 M KCl 0 1 M EDTA 5 mM PMSF 1 mM Table 6 Isofocalisation buffer I Urea 7M Thiourea 2M Triton X 100 0 1 v v CHAPS 4 w v TBP 2 mM Table 7 Isofocalisation buffer I Urea 7M Thi
77. t a large EST database An in house Mascot server does not have these limitations but requires the purchase of the licensed Mascot server The current version of Mascot server requires computers to have high speed Intel and AMD processors at least 2GB RAM and 200 GB IDE hard drive Mascot currently does not support other CPU processors After installing the software one can then create the custom database a Database Maintenance 1 Database maintenance is not a routine task and only performed when you have a new sequence database or new sequences to add to the current local sequence database To create a new Mascot searchable sequence database go to Programs click Mascot The local mascot homepage will appear Click the Database Maintenance button and the database maintenance window comes up as shown in Figure 16 E Mascot Database Maintenance Microsoft Internet Explorer File Edit View Favorites Tools Help e gt 0 Ala a BG B 46 B E Back Forward Stop Refresh Home Search Favorites History Mail Print Edit Discuss Address e http qstar mascot x cgi db gui pl 5 J Go Links Mascot Database Maintenance New database Edit parse rules Edit options Current database definitions Name MsDB Active Inactive Path d INETPUB MASCOT sequence MSDB currenyMSDB_ fa AA C NAC Dio Mem map lv Memlock M Blocks Threads 2 Localreffile V Taxonomy source MSDB REF Rule to parse access
78. ted activation of cell division and embryogenic cell formation but does not influence cell cycle progression in alfalfa cell cultures Plant J 43 849 860 Proteomics page 57 of 62 Medicago truncatula Handbook version March 2007 Perkins D N D J Pappin D M Creasy and J S Cottrell 1999 Probability based protein identification by searching sequence databases using mass spectrometry data Electrophoresis 20 3551 67 Prime T A D J Sherrier P Mahon L C Packman and P Dupree P 2000 A proteomic analysis of organelles from Arabidopsis thaliana Electrophoresis 21 3488 99 Rabilloud T 2002 Two dimensional gel electrophoresis in proteomics old old fashioned but it still climbs up the mountains Proteomics 2 3 10 Ramagli L S and L W Rodriguez 1985 Quantification of microgram amounts of protein in two dimensional polyacrylamide gel electrophoresis sample buffer Electrophoresis 6 559 563 Rose J K S Bashir J J Giovannoni M M Jahn and R S Saravanan 2004 Tackling the plant proteome practical approaches hurdles and experimental tools Plant J 39 715 33 Rossignol M 2001 Analysis of the plant proteome Curr Opin Biotechnol 12 131 4 Saalbach G P Erik and S Wienkoop 2002 Characterisation by proteomics of peribacteroid space and peribacteroid membrane preparations from pea Pisum sativum symbiosomes Proteomics 2 325 337 Sambrook J and D W Russel 2001 Molecular cloning a laboratory manu
79. ted or not with micro organisms To sample roots from plants that have been grown in a soil consisting of any kind of substrate the roots are carefully removed from the soil mix by immersing pots in tap water Then roots are gently rinsed to eliminate any remaining soil particles first with running tap water and secondly with deionized water At this stage part of the root system may be checked for microbial infections i e mycorrhizal bacterial pathogenic nematode infections etc with an appropriate test The remaining root system is weighed and either immediately frozen in liquid nitrogen and stored at 80 C until protein extraction or directly submitted to the selected protein extraction process Products and Buffers It is strongly recommended to use high grade quality electrophoresis products Extraction buffer see Table 3 Biophenol pH 8 0 Tris buffered Biosolve or any other chemical company Cold 0 1 M ammonium acetate in methanol kept at 20 C Cold methanol and cold acetone kept at 20 C Solubilization buffer Table 4 modification of O Farrell s lysis buffer O Farrell 1975 Extraction of total and soluble proteins This protocol has been adapted from Bestel Corre et al 2002 as following 1 Crush roots in liquid nitrogen N2 Routinely 1g fresh weight of M truncatula roots is crushed in a mortar cooled with liquid N2 Once the root sample has become a very thin powder transfer it into
80. teins 2 ul silver stain 15 ul for Commassie if using Mark 12 markers onto a paper wick Let the wick air dry for 5 minutes and coat with 1 agarose 5 Place IEF strip on top of a 1 5 mm thick 10 acrylamide bis or acrylamide PDA gel i 10 acrylamide bis Gel per 12 gels 30 acrylamide bis 396 0 mL 1 5M Tris pH 8 8 300 0 mL Proteomics page 28 of 62 Medicago truncatula Handbook version March 2007 Water 486 0 mL 10 SDS 12 0 mL 10 APS 9 0 mL TEMED 0 9 mL i Place 1X run buffer 25mM Tris 192 mM glycine 0 1 SDS pH 8 3 on top of the gel to help prevent bubble formation at the strip gel interface Note 10X running buffer can be purchased Bio Rad and diluted prior to analysis ii Keep strips wet with equilibration buffer to facilitate their sliding into place iv Once the strip is in place remove excess run buffer by tilting gel cassette and wick with Kimwipe or pipette v Place the marker protein wick onto the gel next to but not touching the Strip 6 Seal strip in place by overlaying with 1 agarose in 1X running buffer containing 0 001 Bromophenol blue Note 1 agarose solutions need to be warmed prior to overlaying This is commonly done by heating the solution in a microwave or on a warming plate 7 Run gels at 25 mA per gel or per the manufacturer s recommended voltage amperage settings 8 When dye front is 1 cm from bottom of the gel 15 5 hours stop the electrophoretic separation Remove the
81. tryptic digestion The resultant data is then compared to theoretical peptide fragments for protein sequences housed in databases using the search engine MASCOT for protein identification The nano LC system consists of an autosampler Famos a valve switching device Switchos and a gradient pump system Ultimate Peptides are first loaded onto a trap column on Switchos for desalting and concentrating The trap column is then switched in line with the analytical column by the Switchos for the separation of peptides Peptides eluting from the column are electrosprayed directly onto the QSTAR and positive ions analyzed by both MS and tandem MS analyses Figure 13 Proteomics page 44 of 62 Medicago truncatula Handbook version March 2007 A waste pump 2 1 149 trap 3 9 Ultimate column 4 8 Gradient syringe 5 6 L Pump sample waste Famos analytical Switchos Ultimate column HV QSTAR B pump 3 Ultimate column i 4 Gradient syringe 56 Pump M sample waste Famos analytical Switchos Ultimate column HV QSTAR Figure 13 Configurations of nano LC system A In load position peptides are transported to the trap column for desalting and concentration The trap column is not in line with the analytical column B In separation position the trap column is now in line with the analytical column for separation 1 Degas solvents solvent A 5 acetonitrile with 0 1 formic acid solvent B 95 acetonitrile with 0
82. uge 6 you will get a four phase system supernatant phenolic phase solid interphase aqueous phase and bottom precipitate Proteomics page 22 of 62 Medicago truncatula Handbook version March 2007 7 remove the supernatant phenolic phase to a pre chilled and clean tube preferably 50ml Falcon tube 8 do not include the interphase or aqueous phase Table 13 Denaturing Extraction Buffer DEB 50mM HEPES KOH pH7 5 40 Sucrose 1 B Mercaptoethanol 60mM Sodium fluoride ImM EDTA sodium salt ImM Sodium orthovanadate ImM Benzamidine 50uL 10mL Sigma Plant Protease Inhibitor Cocktail 0 3uM Mikrocystin solubilise HEPES and sucrose first adjust pH add inhibitors and fill up to desired volume Variations In case you expect a lot of interfering compounds eg seeds you can wash the phenolic phase with additional 2 5ml DEB for 10 min at 4 C or you can also increase the amount of DEB Buffer in the first step use 5ml instead of 2 5ml Depending on the certain material you need to try out what works best Acetone Precipitation l precipitate proteins out of the phenolic phase by adding five volumes 30 40mL of ice cold acetone at 20 C overnight 16h centrifuge at 4 C and 3 250g for 10min remove the supernatant crush the pellet with ice cold methanol the pellet is very sticky and chewy centrifuge at 4 C and 3 250g for 10min wash the pellet for a total number of 2 3 washing steps dry for 20min at room temperatu
83. utes Set sonicator Model 150 V T ultrasonic homogenizer Biologics Inc to Power Output 50 and Pulse 70 ii Repeat a and b for a total of two times 5 Centrifuge at 5 000 g for five minutes 6 500 rpm in SM 24 rotor to pellet debris and recover the supernatant 7 8 mL 6 Precipitate protein by adding 4 33 mL of 37 5 TCA plus 1 0 D mercaptoethanol bring to a final volume of 13 mL using 37 5 TCA incubate on ice for 45 minutes and centrifuge at 14 000 g for 15 min 7 Wash protein pellet 3 times in 80 acetone containing 0 05 D mercaptoethanol 1 Break up protein pellet using small metal spatula li Sonicating in a water bath for five minutes will help disrupt the pellet Avoid heating of samples by checking intermittently and adding ice to the sonicator bath if necessary 8 Aur dry protein pellet in laminar flow hood until all the liquid is gone but the pellet still looks damp Store protein samples at 80 C Over drying the pellet will cause problems lower recovery in the solubilization step Proteomics page 5 of 62 Medicago truncatula Handbook version March 2007 2 2 Green tissues Differentiated green tissues leaves are typically more problematic during 2 DE analysis than cell cultures because they contain high levels of salts chlorophyll and other molecules polyphenolics polysaccharides lignins pigments etc that interfere with isoelectric focusing The relatively large abundance of ph
84. veloping solution D Silver Developing Solution 3g sodium carbonate 2mL Sensitising Solution 50uL formaldehyde in 100mL ddH O page 26 of 62 Medicago truncatula Handbook version March 2007 3 Protein separation 3 1 2D gel electrophoresis a Resolubilization rehydration and isoelectric focusing i e 1 dimensional separation 12 Samples Lei et al 2005 1 Rehydrate pellets in 1 mL of IEF solubilization buffer containing 9 M Urea 3 CHAPS 2 Triton X 100 20 mM DTT 1 ampholytes i IEF solubilization buffer per 17 5 mL Urea 9 45 g CHAPS 525 mg Triton X 100 350 uL DTT 54 mg Ampholytes 175 uL i Break up protein pellet using small metal spatula ii Degas protein solution in water bath sonicator for five minutes Be sure the sample doesn t heat up while in the sonicating water bath iv Place the samples on orbital shaker set to slow speed for hour v Vortex occasionally to assist resolubilization 2 Transfer to 1 5 mL microfuge tube 1 Break up protein pellet with a blue pestle Alternatively sonicating in a water bath for five minutes will help disrupt the pellet ii Ifusing a pestle ensure that no protein chunks stick to the pestle 3 Degas in water bath for 5 minutes followed by sonication for 30 minutes Check and prevent heating of the samples by adding ice to the sonicator bath 4 Allow proteins to solubilize for at least an hour with occasional mixing 5 Pellet insoluble material
85. wman Keuls test using the SAS software package SAS Institute 1999 Table 19 Experimental characteristics of the protein spots identified by mass spectrometry and used as internal references to normalize spot volumes across the different gels Tentative A Mr Putative protein name consensus sequence from pl kDa TIGR Molecular chaperone Hsp90 2 TC85438 5 0 89 Heat shock 70 kDa protein TC85718 4 6 80 Type IIB calcium ATPase MCA5 TC85483 5 8 83 F1 ATPase TC85540 5 3 58 Putative TPR repeat protein TC91689 5 8 75 Putative aminoaldehyde dehydrogenase TC77827 5 4 60 RuBisCO subunit binding protein alpha TC85910 27 62 RuBisCO subunit binding protein beta TC76847 5 3 64 two spots 5 3 65 Glucose 6 phosphate isomerase TC77538 5 9 64 The Institute for Genomic Research http www tigr org Proteomics page 35 of 62 Medicago truncatula Handbook version March 2007 4 3 Difference Gel Electrophoresis DIGE One major drawback of comparative proteomics by 2D gel analysis is the matching of 2D gels A recent technique to improve matching of gels from different samples is DIGE Difference Gel Electrophoresis Unlii et al 1997 Figure 7 This technique is designed to make it easier to compare gels of different biological samples for example a mutant and wild type tissue M vs WT The main difference conventional 2D gel electrophoresis is that the two samples to be compared are run on the same gel thereby the same protein from each
86. xample phenolics and flavonoids We have found that extraction preceded by a precipitation method with tricholoacetic acid TCA and acetone leads to a clean protein preparation The protein yields are approximately 0 1 of fresh weight in roots and up to ten times more in less vacuolated tissues like root tips and nodules A typical protein extraction protocol will take about a day 1 Harvest tissue quickly into liquid nitrogen to prevent and protein breakdown This is particularly important if you want to look at post translational modifications for example phosphorylation see 2 9 Store at 80 C until needed 2 Prepare the following solutions on the day of the extraction and store at 20 C e 10 TCA in acetone including 0 07 w v dithiothreitol DTT e 100 acetone including 0 07 w v DTT 3 Prepare Solubilization Buffer Table 2 on the day or beforehand and keep frozen at 80 C for up to 3 months until the day of the extraction Table 2 Solubilization buffer Ingredient Add for a final Final Comments volume of 10ml concentration Urea 5 4g 9 M Solubilizes proteins DTT 0 1 g 1 w v Breaks S S bonds CHAPS 0 4 g 4 w v Detergent Biolyte 100 uL Help disaggregating Amphotytes proteins pH 3 10 Tris base 0 042 g 35 mM Buffer EDTA 100 uL of 05M SuM Protease inhibitor stock removes Ca ions PMSF 100 uL of 100 1 uM Protease inhibitor mM stock in very toxic ethanol Purified water Up to 10 ml Additional com
87. ysis via nano LC QTOF MS MS 5 3 Database searching 6 Concluding remarks 7 Abbreviations 8 References Proteomics page 2 of 62 Medicago truncatula Handbook version March 2007 Abstract Proteomics has evolved greatly in the past few years and the number of researchers utilizing proteomics to investigate plant biology has substantially increased The predominant aim of this chapter is to aid Medicago researchers in initiating proteomics experiments The chapter consists of a brief literature review of Medicago proteomics followed by protocols used in the authors labs Further optimization of the protocols to suit ones individual needs may be required 1 Introduction to proteome analysis Legumes particularly soybean and alfalfa are economically important crops with an estimated US annual value of 17 billion and 7 billion respectively USDA NASS 2005 Though substantial research is being conducted using soybean and alfalfa Medicago truncatula has gained popularity as a model legume because of its small genome size self fertility rapid generation time large number of mutants large expressed sequence tag libraries and genomic sequencing Bell et al 2001 Cook 1999 Trieu et al 2000 Both custom and commercial transcriptome based approaches have been established in Medicago research See chapter Transcriptomics however transcript profiles do not always provide a complete story due to limited correlations in transcript and

Proteomics of Medicago truncatula

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