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USER`S MANUAL

1. Tip Ficure 4 Wet the sorbent with Eluting Medium and stop before it gets to the filter then eject the liquid and sorbent particles out of the tip Figure 4 15 Transfer part of the upper organic layer about 50uL into an autosampler vial included using a Pasteur pipette Avoid the transfer of aqueous layer along with the organic layer Evaporate to dryness in a gentle stream of nitrogen at room temperature Do not leave samples in the nitrogen stream for more than 10 minutes Re dissolve in 70 100 uL mixture of HPLC mobile phase components 10mM ammonium formate in water 10mM ammonium formate in methanol 1 2 v v Transfer reconstituted sample into an insert included and place insert into the same autosampler vial The sample is ready for LC MS analysis 3 5 Optimizing Sample Preparation Time For experienced users sample preparation proceeds in 7 8 minutes per sample This pro cess can be further improved by preparing up to ten samples at a time For example at step 2 dispense Reagent 1 and at later steps all other reagents in ten vials successively using the same pipette tip At step 9 after dispensing Reagent 4 vortex 2 3 vials simultaneously During each 1 minute wait at steps 10 12 prepare autosampler vials for sample transfer EZ faast User s Manual 4 0 LC MS Anatysis 4 1 Column For EZ faast LC MS Analysis The HPLC column included in the kit should be based on the flow rate most compatible
2. gt breaking with tradition in Ca Rc 1x REF Phenomenex Ltd Deutschland Zeppelinstr 5 63741 Aschaffenburg Deutschland of Protein Hydrolysates by LC MS For Part Numbers KHO 7339 KHO 7340 phenomenex breaking with tradition 411 Madrid Ave Torrance 90501 1430 USA l 2 E P 2 E m EL 2 5 C V N EZ faast User s Manual The following is a description of the symbols used in the EZ faast manuals on EZ faast packaging and on EZ faast kit components Symbol for In Vitro Diagnostic Medical Device Symbol for Manufacturer Symbol for Authorised Representative In The European Community ud T Symbol for Use By and or Expiration Date 9 Symbol for Batch Code and or Lot Number Symbol for Catalogue Number Symbol for Serial Number Symbol for Flammable Substances Symbol for Irritating or Harmful Substances oh Me Symbol for Corrosive Substances TABLE OF CONTENTS maaa 1 2 Sample Preparation 5 om aaa 12 Sample Storage and Stability 16 Cleaning
3. and place them in the freezer For longer storage we recommend that the organic layer be desiccated with anhydrous sodium sulfate before solvent removal vials be capped and placed in the freezer Since sample preparation is expeditious with this procedure we recommend analyzing samples prepared freshly Samples prepared during the day may be left on the autosampler tray at room temperature to be analyzed during the night or the next day 6 0 CLEANING AND CARE or SUPPLIES The Dialamatic Microdispenser should be flushed with isopropanol acetone approx 1 1 at the end of the day Please review the Drummond Microdispenser users manual for further care and use notes The same organic mix is recommended as wash for both manual syringes and autosamplers Plastic syringes used for SPE can be cleaned by flushing with a propanol water 1 2 v v mixture Always tightly cap the reagent bottles when not in use in order to avoid solvent evapora tion and alteration of reagent composition Cover the racks holding sorbent tips when not in use to prevent contamination EZ faast User s Manual 7 0 Quality ASSURANCE All components of the EZ faast Amino Acid Analysis kit are subjected to rigorous quality control testing These measures help to ensure the best results If poor results occur please contact your Phenomenex technical consultant or distributor 8 0 Propuct Limitations Phenomenex Analyte Specific Reagent pro
4. 234 3 146 0 amu Period 1 204 1 144 0 amu Period 1 248 3 160 2 amu 3 71 4 25 4 32 15e5 SER 9 7e5 GLY 13066 THR 0 0 1 2 3 1 2 3 4 1 2 3 4min p of MRM 11 of MRM 11 gt _ XIC of MRM 11pairs Period 2 218 3 130 1 amu Period 2 377 0 317 0 amu Period 2 281 1 193 2 amu 514 43 6 94 2 9 6 6 9e6 1 176 ALA HLY IS d3MET 8 48 0 L 1 4 0 Z EZ faast User s Manual gt of MRM 11pairs Period 2 278 3 190 1 amu 7 04 4 li MET min gt of MRM 11pairs Period 2 304 3 216 2 amu 7 76 4966 ASP XIC of MRM 11pairs Period 2 318 3 172 1 amu 8 33 2566 GLU P XIC of MRM pairs Period 3 308 3 220 2 amu 12 13 3 8e6 IS HPHE 13 min D gt of MRM 11pairs Period 2 244 3 156 2 amu 7 13 p XIC of MRM 1 1pairs Period 2 361 2 301 3 amu 7 72 7 0e6 PRO 5 0e6 LYS 0 2 5 6 7 8 min gt XIC of MRM 11 pairs of MRM 1 1pairs Period 2 370 2 196 3 amu Period 2 246 3 158 1 amu 7 82 820 4066 HIS 3 666 VAL 7 23 gt 0 0 5 6 7 8 min 5 0 6 0 7 0 8 0 min of MRM 11pairs of MRM 5pairs Period 2 333 3 245 1 amu Period 3 260 3 172 1 amu 8 49 10 01 4 06614 TRP 5 0e6 ILE LEU 10 54 gt ____ 0 5 6 7 8 min 10 11 12 13 min XIC of MRM 5pairs of MRM
5. and Care of Supplies 16 17 Product 18 Ordering Information 19 20 EZ faast User s Manual 1 0 Kit Components 1 1 Reagents Reagent Ingredients Volume Reagent 1 Homoarginine 0 2 mM 50mL Internal Standard Solution Methionine d 0 2 mM Homophenylalanine 0 2 mM Reagent 2 Na CO 90mL Sodium Carbonate Solution Reagent 3A Sodium Hydroxide 60mL Eluting Medium Component I Reagent 3B N propanol 40mL Eluting Medium Component II Regent 4 Chloroform 4 vials 6mL each Organic Solution Reagent 5 Iso octane 50mL Organic Solution II SD Please refer to section 4 6 in 2 vials 2mL each Protein Amino Acid Standard the manual Mixtures 1 2 Supplies e Sorbent tips in racks 4x96 Sample preparation Vial 4x100 Microdispenser 20 100IIL n n 1 Syringe 0 6mL Syringe 1 5mL EZ faast AAA HPLC Column nasa 1 Autosampler vials with inserts 4x100 User SENA EZ faast Demo Video and Reference CD sss 1 1 3 Materials Required but Not Supplied In Kit e 100uL 1mL pipette SoftGrip pipette Phenomenex P N AH0 5968 or equivalent 30 300uL pipette SoftGrip pipette Phenomenex P N 5967 or equivalent 10 10001 pipette SoftGrip pipette Phenomenex P N 5966 or equivalent Pipette tips Ph
6. and demo video Description GC FID Free Physiological Amino Acid Analysis Kit GC MS Free Physiological Amino Acid Analysis Kit GC FID Protein Hydrolysate Kit GC MS Protein Hydrolysate Kit LC MS Free Physiological Amino Acid Analysis Kit with 250 x 2 0mm column LC MS Free Physiological Amino Acid Analysis Kit with 250 x 3 0mm column LC MS Protein Hydrolysate Kit with 250 x 2 0mm column LC MS Protein Hydrolysate Kit with 250 x 3 0mm column GC Free Physiological Amino Acid Standards SD1 SD2 amp SD3 2mL vial x 2 GC Protein Hydrolysate Standard SD 2mL vial x 2 LC MS Free Physiological Amino Acid Standards for LC SD1 SD2 amp SD3 2mL vial x 2 LC MS Protein Hydrolysate Standard SD 2mL vial x 2 Order No KG0 7165 KG0 7166 KG0 7167 KG0 7168 KH0 7337 KH0 7338 KH0 7339 KH0 7340 AGO 7184 AGO 7263 ALO 7500 ALO 7501 Unit ea ea ea ea ea ea ea ea ea ea ea ea Phenex Vials This universal vial can be used in any autosampler that utilizes a 12 x 32mm vial It may be used in place of crimp top and snap ring top vials Eliminates the need of stocking many different style vials The top screws down in 1 3 turn and eliminates the chore of crimping de crimping and snapping caps on Cap comes with a bonded in septa that eliminates septa slipping into vials Vials and caps with bonded in septa come in one convenient kit pack Description Order No Clear wide mouth vial cap and septa kit pack wi
7. are numerous published methods for protein hydrolysis all are compatible with analysis by the EZ faast procedure with minor modifications to the described method The most common methods use acid hydrolysis with 6M HCI in either liquid or vapor phase Stein and Moore Methods in Enzymology 6 819 831 1963 Tarr et al In Microcharaterization of Proteins J E Shively ed Humana Press 1986 While these methods give good results for a majority of amino acids there are several amino acids that are either partially or completely destroyed by such methods and alternate hydrolysis methods must be used For convenience a common liquid and vapor phase method are described hydrolysis reagents and supplies are not included with the EZ faast kit EZ faast User s Manual 3 3 2 Vapor Phase Hydrolysis The following is a sample method for vapor phase hydrolysis as a reference other meth ods may work better for your application 1 Transfer 1 20 nanomoles of protein into an autosampler vial insert 2 Lyophilize sample in a vacuum concentrator 3 Ina hydrolysis vessel add 989yL 6N constant boiling HCI 10 5 Phenol and 1pL betamercaptoethanol 4 Add vial inserts into hydrolysis vessel and cap with minaret valve 5 Place vessel in an ice bucket and purge with nitrogen and vacuum several times and seal vessel under vacuum 6 Hydrolyze in oven at 110 C for 24 hours 7 Cool vessel and remove vial inserts 8 Dry do
8. eluting amino acids MET d3 as the internal standard for SER through GLU TRP middle eluting amino acids and HPHE for LEU through TYR late amino acids Remember the SD vial should be placed in the freezer after use Allow standards to reach room temperature before use Extracted lon Chromatograms for protein amino acids and for the internal standards included in Reagent 1 are shown below 4 6 Calculation of Analytical Results Calculation of amino acid levels in hydrolysed samples are performed by the Data Analysis portion of the software controlling the analytical instrument liquid chromatograph Calcula tions and calibration are based on the internal standard s Results are reported in the units entered for internal standard s and analyte levels in calibration mixtures Note nmols mL are equivalent to pmols L EZ faast User s Manual Extracted ion chromatograms for the amino acids included in the standard mixture pro vided with the kit in order of elution at 200nmol mL each XIC of MRM 12pairs Period 1 303 4 69 9 amu gt of MRM 12pairs Period 1 317 3 84 1 amu gt of MRM 12pairs Period 1 260 2 172 1 amu 7 3e5 a 1 00 6 E i 196 ARG 00 15 5 0e6 4 29 0 T T T A T T T 0 T T 1 2 3 4 min 1 2 3 4 min 1 2 3 4 min b gt of MRM 12pairs gt of MRM 12pairs gt _ XIC of MRM 12pairs Period 1
9. stored at room temperature For your convenience the bottom of the reagent box has been designed as a tray that can be easily lifted from the workstation and placed in the refrigerator when the kit is not in use for an extended period of time All components are guaranteed for 12 months or more see label on bottle vial from the date of purchase when stored at recommended temperatures and used as described in this manual Please review the Instruction Manual included with the Drummond Dialamatic Microdispenser for recommended usage and warranty information Please observe recom mendations for solvent bottle handling and syringe cleaning in Section 6 0 of this manual 2 4 Safety Although the concentration of all toxic components in any of the reagent bottles is low for safety reasons the sample preparation station should be placed in an exhaust hood and protective gloves and goggles should be worn When working with biological fluids please take any necessary precautions to prevent infection with blood borne pathogens Appropriate bio safety precautions and disposal of bio hazardous wastes should be followed 3 0 PREPARATION PROCEDURE 3 1 Setup The EZ faast kit packaging has been designed as an efficient workstation It holds a reagent tray a vial rack a pipette rack and a section for sorbent tips and vials To speed up sample preparation it is recommended that the workstation be arranged as shown in figure 1a By following
10. with your LC MS system Flow 0 5 mL min EZ faast AAA MS column 250 x 3 0 mm Flow 0 25mL min EZ faast AAA MS column 250 x 2 0 mm Column should be equilibrated by running a blank gradient Column can be stored in mobile phase when not in use Note because of column length and the use of a sorbent with small particle size and a mobile phase of high viscosity methanol water the expected column backpressure is 200 220 bar 2 900 3 200 psi The column Supplied with the kit will tolerate this backpressure very well 4 2 Instrument Settings LC Mobile phase 10mM Ammonium formate in water B 10mM Ammonium formate in methanol Gradient 00 00min 68 B 13 00 83 B 13 01 68 B 17 00 68 B Re equilibration time may vary between 4 and 7 minutes depending on the gradient delay time of the HPLC instrument Flow rate 0 50mL min for 3 0mm ID column 0 25mL min for 2 0mm ID column Column temperature 35 C Injection volume 1 5 Either ESI or be used Mode Positive lon Scan range 100 600 m z ESI ion source temperature 365 C Bruker 425 C AB API 3000 ionization chamber temperature 450 C EZ faast User s Manual 4 3 Tuning the Mass Spectrometer Some mass spectrometers require a concentrated calibration solution for tuning the instrument if not then calibration solution III see section 4 5 can be used To prepare the concentrated solution dis
11. 01625 501796 44 1625 501796 06021 58830 11 09 4780952 02 9428 6445 email info info info franceinfo ukinfo eireinfo anfrage info info phenomenex com phenomenex com phenomenex com phenomenex com phenomenex com phenomenex com phenomenex co nz phenomenex com au
12. 5pairs Period 3 294 3 206 1 amu Period 3 497 2 248 2 amu 12 44 10 10 5 PHE 5 0e6 C C O qo 11 12 13 min 12 13 min EZ faast User s Manual gt XIC of MRM 5pairs TIC from Sample 3 SDLC 1001006 of SDLC 100yl SET6 wiff Turbo Spray Period 3 396 2 136 2 amu Smoothed 13 29 Period 1 Period2 7 76 Period3 2 5 6 TYR 11667 TIC 10 08 713 Aan 6 94 8 49 4 24 5 15 2 75 2 87 Lalo 1 2 3 4 5 6 7 8 9 10 11 12 13 min 10 11 12 13 min 4 7 Amino Acid and Protein Quantitation Calculations For additional information regarding protein quantitation calculations as well as example calculation spreadsheets please refer to reference CD included in kit 5 0 STORAGE AND STABILITY Some amino acids are chemically unstable in physiological fluids e g progressive decline of plasma glutamine and cystine in time and also in standard mixtures Keep samples and standard mixtures in the freezer Old amino acid standard mixtures and mixtures which have not been stored properly should not be used for instrument calibration Order fresh mixtures see ordering info on page 19 of this manual Samples prepared for LC MS analysis following the procedure outlined in this manual may be stored for several days in a freezer before analysis Storing dry samples is preferable to storing reconstituted samples Dry down the organic solvent as described in section 3 4 step 15 cap vials
13. GC Protein Hydrolysate Standard SD 2mL vial x 2 LC MS Free Physiological Amino Acid Standards for LC SD1 SD2 amp SD3 2mL vial x 2 LC MS Protein Hydrolysate Standard SD 2mL vial x 2 Order No KG0 7165 KG0 7166 KG0 7167 KG0 7168 KH0 7337 KH0 7338 KH0 7339 KH0 7340 AGO 7184 0 7263 ALO 7500 ALO 7501 Unit ea ea ea ea ea ea ea ea ea ea ea ea www phenomenex com amp K 7 A e y Sa E Phenomenex products are available worldwide For the distributor in your coi milk ka phenomenex contact Phenomenex USA International Department by telephone fax or iternational phenomenex com breaking with tradition USA Puerto Rico Canada France United Kingdom Ireland Germany New Zealand Australia mail 411 Madrid Ave 273 Sierra Morena 411 Madrid Ave Parc des Grillons Bat3 Queens Avenue Queens Avenue Zeppelinstr 5 P 0 Box 31601 PO Box 4084 Torrance CA Suite 104 Torrance CA 60 route de Sartrouville Hurdsfield Ind Est Hurdsfield Ind Est 63741 Aschaffenburg Milford Lane Cove NSW 2066 90501 1430 San Juan 90501 1430 78232 Le Pecq Cedex Macclesfield Cheshire Macclesfield Cheshire Germany Auckland Australia USA Puerto Rico 00926 USA France SK10 2BN UK SK10 2BN UK New Zealand tel 310 212 0555 800 541 800 543 3681 01 30 09 21 10 01625 501367 01 247 5405 06021 58830 0 09 4780951 02 9428 6444 fax 310 328 7768 310 328 7768 310 328 7768 01 30 09 21 11
14. Z faast User s Manual Table 2 The comprehensive list of amino acids and related compounds prepared by EZ faast for LC MS analysis internal standards listed in bold Ethanolamine 2 7 611 148 62 88 106 Pyroglutamic acid pGLU 2 6 129 1 172 130 Arginine ARG 2 7 174 2 303 70 156 286 2 Homoarginine HARG 29 188 2 317 84 128 170 NA Glutamine GLN Q 3 3 146 2 275 172 84 215 5 Citrulline CIT 3 4 175 2 304 156 113 287 Anserine ANS 3 5 240 1 369 309 212 2 Methionine sulfoxide 3 5 165 2 294 234 142 Serine SER 5 3 7 105 1 234 146 174 216 ASN N 3 8 1321 243 157 115 201 10 Proline hydroxyproline 3 8 228 2 357 156 184 297 dipeptide Theanine THE 3 8 1742 303 243 215 3 Methyl histidine 3MHIS 3 8 169 2 298 210 256 5 1 Methyl histidine 1MHIS 3 8 169 2 298 96 196 5 4 Hydroxyproline HYP 4 131 1 260 172 157 200 5 Glycine GLY G 43 751 204 144 118 162 5 Glycine proline dipeptide GPR 43 1722 301 158 241 1 Threonine THR T 43 119 1 248 160 188 230 10 Methionine sulfone 44 1812 2310 250 222 142 Alanyl alanine dipeptide ALA ALA 45 160 2 289 229 Methionine sulfoximine 5 180 2 395 230 188 p Alanine pALA b 89 1 218 98 116 158 Alanine ALA A 5 1 89 1 218 130 158 88 20 6 Hydroxylysine HLY 54 1621 377 317 125 359 100 4 Aminobutyric acid GABA 5 6 103 1 232 172 130 B5 Hi
15. alanine PHE F 10 1 165 2 294 206 120 1 allo Isoleucine alLE 10 5 131 2 260 172 200 Isoleucine ILE 10 6 131 2 260 172 130 74 5 Adrenaline 10 6 183 2 424 338 382 252 Norleucine NLEU 10 9 131 2 260 172 200 Cysteine CYS 10 9 121 2 336 190 248 276 Aspartame acid ASP PHE 11 2 280 3 451 391 Aminopimelic acid APA 11 4 175 2 346 258 198 286 5 Arginino succinic acid 11 4 334 2 529 383 443 Pipecolic acid HPRO 11 5 129 1 258 170 198 128 Dopamine DA 11 5 153 1 412 266 326 Lysinoalanine LAL 11 7 233 2 430 370 342 Cystathionine CTH 11 9 222 3 479 230 188 419 5 4 Aminobenzoic acid PABA 11 9 137 1 266 224 Homocysteine HCYS 12 1352 350 204 290 Homophenylalanine HPHE 12 2 179 2 308 220 117 104 NA Cystine C C C 12 5 240 3 497 248 437 306 5 Tyrosine TYR Y 13 3 181 2 396 136 308 336 10 Cysteine Homocysteine C HC 13 3 320 1 511 262 451 dipeptide 3 Hydroxy tyrosine DOPA 13 5 197 2 498 352 412 438 Homocystine HC CH 13 8 268 3 525 262 88 465 Seleno cystine Se C 14 3 3341 593 296 371 Dimethylarginine SDMA 10 0 202 2 331 98 Dimethylarginine ADMA 1129 202 2 331 98 1008 were determined for amino acids included in the standard mixtures provided with the kit extended gradient time required alternate chromatographic conditions required EZ faast User s Manual 2 3 Storage and Stability Store Reagents 1 3B and 4 at 4 C Store amino acid standard solutions in the freezer All other components may be
16. directions and markings on the reagent box by breaking it along perfora tions it can be transformed into a reagent tray When the kit is not in use for several days the reagent tray figure 1b may be conveniently removed and placed in the refrigerator WorksTATION ARRANGEMENT FicunE 1 To speed up sample preparation it is recommended that the workstation be arranged as shown below Figure 1a Figure 1b EZ faast User s Manual 3 2 Preparing the Eluting Medium The volume of prepared Eluting Medium depends upon the number of samples to be ana lyzed during the day 200uL sample The eluting medium should be prepared fresh each day 1 Use capped vials of appropriate size not included for preparation of the Eluting Medium 2 Combine 3 parts Reagent 3A Eluting Medium Component with 2 parts Reagent 3B Eluting Medium Component Il in an appropriate sized vial see Table 3 page 6 for reagent volumes based on number of samples Mix briefly 3 Store prepared eluting medium during the day at room temperature Discard any unused mixture at the end of the day Table 3 For your convenience check the table below to determine the volume of Eluting Medium components needed depending on your number of samples 600pL 7 900pL 12 1 5mL 14 1 8mL 19 2 4mL 24 3 0mL 29 3 6mL 34 4 2mL 39 4 8mL 44 5 4mL 49 6 0mL 3 3 Protein Sample Hydrolysis 3 3 1 Background There
17. ducts are not intended for clinical use Because they are not intended for clinical use no claim or representation is made or intended for their Clinical use including but not limited to diagnostic prognostic therapeutic or blood banking It is the user s responsibility to validate the performance of Phenomenex products for any par ticular use since the performance characteristics are not established Phenomenex products may be used in clinical diagnostic laboratory systems after the laboratory has validated their complete system as required by the Clinical Laboratory Improvements Amendments of 1988 88 regulation in the U S or equivalent in other countries Trademarks EZ faast Sorbent Tips are patented Phenomenex Inc U S Patent 6 770 246 EZ faast is a trademark of Phenomenex Inc Phenex is a trademark of Phenomenex Inc FocusLiner is a trademark of SGE SoftGrip is a trademark of Hamilton Drummond is a registered trademark of the Drummond Corp Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by Law 2005 Phenomenex Inc All rights reserved EZ faast Kit Each kit includes a EZ faast AAA LC column or ZB AAA GC column and liners sample prep and derivatization reagents sample prep vials AA standard mixtures SPE sorbent tips autosampler vials with inserts come with MS kits Microdispenser for Reagents 4 and 5
18. e Sample Preparation by SPE and Derivatization procedure described in this manual in Section 3 4 With standard samples no pH adjustment is necessary Just add 100uL of Reagent 1 Internal Standards Solution and proceed with sample preparation The protein hydrolysate amino acid standard mixture SD is composed of the following amino acids 200nmoles mL each ALA GLU HYP MET THR ARG GLY ILE PHE TRP ASP HIS LEU PRO TYR C C HLY LYS SER VAL TRP can be analyzed only in hydrolysates prepared under alkaline conditions Note the amino acids included in SD are the most widely analyzed for protein hydrolysates For assistance with additional amino acids in your hydrolysate sample please contact Phenomenex 4 5 Calibration Procedure Use the following standard amino acid mixtures and make duplicate injections of each to generate the desired calibration Calibration Solution 1 1001 of SD solution plus 100pL Internal Standard solution Calibration level 20 nmol mL 5 SD solution plus 100uL IS Calibration level II 100 nmol mL 100pL SD solution plus 100pL IS Calibration level Ill 200 nmol mL The concentration of each internal standard IS HARG MET d3 and in calibrators and samples prepared for chromatographic analysis is 200 nmoles mL While the use of the ideal internal standard will vary based on instrument and application we recommend using HARG as the internal standard for ARG and other early
19. enex Phenomenex P N 5917 200uL and 5920 1mL or equivalent Vortex Vials of an appropriate volume with caps see section 3 2 Pasteur pipettes for sample transfer see section 3 4 step 15 Container for proper waste disposal Reagents and supplies for Protein Hydrolysis EZ faast User s Manual 2 0 Overview 2 1 Overview The EZ faast amino acid analysis procedure consists of a solid phase extraction step followed by a derivatization and a liquid liquid extraction derivatized samples are quickly ana lyzed by liquid chromatography mass spectrometry The solid phase extraction is performed via a sorbent packed tip that binds amino acids while allowing interfering compounds to flow through Amino acids on sorbent are then extruded into the sample vial and quickly derivatized with reagent at room temperature in aqueous solution Derivatized amino acids concomitantly migrate to the organic layer for additional separation from interfering compounds Organic layer is then removed evaporated and re dissolved in aqueous mobile phase and analyzed on aLC MS system Total sample preparation time takes around 8 minutes and analysis is performed in around 12 minutes for a total start to finish time of around 20 minutes A video included with this kit demonstrates the simplicity of the procedure Please be aware that some sample preparation steps described in the video may be different than what is described i
20. gent 1 to the amino acid standard mixture and proceed with the SPE as described at step 3 EZ faast User s Manual Grass Line Up Ficure 2 Figure 2 ed 3 For each sample line up one sorbent tip and one glass sample preparation vial in the vial rack Attach a sorbent tip to a 1 5mL syringe and loosen the syringe piston immerse the tip and pass the solution in the sample preparation vial through the sorbent tip by SLOWLY pulling back the syringe piston in steps Caution Do not quickly pull back the piston The syringe should be capable of drawing all sample and subsequent wash into the barrel Watch as the liquid accumulates inside the syringe barrel and move the piston only as the accumulation slows down Try to take at least 1 minute to pass hydrolysate or standard solution samples through the sorbent tip If you run out of piston range detach the sorbent tip expel the solution from syringe barrel then reattach the sorbent tip and proceed with sample preparation Note the sorbent tip should stay in the sample preparation vial through steps 3 10 see Figure 3 even when dispensing reagents In case the sorbent tip cannot reach to the bottom of the vial tilt the vial to 45 and proceed with the SPE step 4 5 Pipette 2001 HPLC grade water into the same sample preparation vial Pass the water through the sorbent tip and into the syringe barrel SLOWLY Drain the liquid from the sorbent bed by pu
21. gments looks as follows Time Suggested Tune AA AA in Range AA at End of Range 0 4 8 min SER and THR R S G T THR 4 8 6 5 min ALA A ALA 6 5 7 7 min HLY and PRO 7 7 9 3 min VAL and ASP K D H V E W GLU or TRP 9 3 10 7 min LEU and PHE ILE 10 7 14 min C C and TYR C C Y TYR The segment time limits and amino acids to tune for may be different depending on instru ment and application HPLC pumps having larger gradient delay times will produce longer retention times and segments must be adjusted accordingly For your convenience we have included methods for Applied Biosystems API3000 LC MS MS and Bruker Daltonics Es quire2000 LC MS instruments on the reference CD included with this kit To use any of these files copy it into the appropriate folder in your software Text file versions of the method files illustrate instruments settings including tune parameters Note With some mass spectrometers sodated molecular ions derivative MW 22 are more prevalent than pro tonated ions e g m z 330 maybe observed for HPHE instead of m z 308 In such cases it is beneficial to acidify the sample with formic acid by reconstituting the sample prepared for tuning in 0 1 formic acid and 10mM ammonium formate in both methanol and water 2 1 v v EZ faast User s Manual 4 4 Calibration Standards For quantitation purposes aliquots of the amino acid standard mixture should be prepared following th
22. ion 3 2 into the sample preparation vial Pull back the piston of a 0 6mL syringe halfway up the barrel and attach the sorbent tip Wet the sorbent with Eluting Medium stop when the liquid reaches the filter plug in the sorbent tip co c A Eject the liquid and sorbent out of the tip and into the sample preparation vial Repeat until all sorbent particles in the tip are expelled into the sample preparation vial Discard the empty tip 11 Using the Drummond Dialamatic Microdispenser transfer 50uL Reagent 4 12 Emulsify by repeatedly vortexing the solution for about 5 seconds Allow reaction to proceed for about 1 minute 13 Vortex the solution again for a few seconds to re emulsify the content of the vial Allow the reaction to proceed for at least one additional minute 14 Using the Microdispenser transfer 100pL Reagent 5 and re emulsify by vortexing for about 5 seconds Let the reaction proceed for 1 minute 15 Transfer part of the upper organic layer 50 100pL using a Pasteur pipette into an autosampler vial Avoid transferring aqueous layer along with the organic layer Evaporate the solvent SLOWLY to dry under a gentle stream of nitrogen max 10 min Stop when sample almost dry Re dissolve amino acid derivatives in 100pL or less of a mixture of LC mobile phase components 1 2 v v Transfer the reconstituted sample into an insert and place the insert in the same autosampler vial The reconstit
23. lling air through the sorbent tip Detach the syringe from the sorbent tip while keeping the tip inside the sample preparation vial Discard the liquid accumulated in the syringe Note save the syringe as it can be reused with many other samples For convenience place it into the pipette rack 7 8 Pipette 200 Eluting Medium prepared fresh each day section 3 2 into the same sample preparation vial Pull back the piston of a 0 6 mL syringe halfway up the barrel and attach the sorbent tip used in steps 3 6 EZ faast User s Manual KEEP THE SORBENT TIP IN THE VIAL FicunE 3 Keep the sorbent tip in the sample preparation vial through steps 3 10 even while dispensing HPLC grade water at step 4 and eluting medium at step 7 Figure 3 9 Wet the sorbent with Eluting Medium watch as the liquid rises through the sorbent particles and stop when the liquid reaches the filter plug in the sorbent tip 10 Eject the liquid and sorbent particles out of the tip and into the sample preparation vial Repeat step 9 and 10 until the sorbent particles in the tip are expelled into the sample preparation vial Only the filter disk should remain in the empty tip see Figure 4 Discard the empty tip Keep the syringe as it can be reused with many other samples Using the adjustable Drummond Dialamatic Microdispenser included transfer 50uL Reagent 4 into the sample preparation vial Caution Avoid cross contamination by n
24. n this users manual Please use the video as a general guide but follow the exact steps and sequence described in this manual 2 2 Amino Acids in Physiological and Protein Hydrolysate Samples The EZ faast method has been developed for the analysis of amino acids from protein hy drolysates Table 1 In total over 60 aliphatic and aromatic amino acids or related compounds can be analyzed with this kit Table 2 Additional amino acids and related compounds may be analyzed with this kit A brief adjustment of chromatographic and MS conditions may be required Please contact your Phenomenex technical consultant for method modifications and other LC and GC amino acid kits Table 1 Protein Amino Acids analyzed by the EZ faast Hydrolysate Amino Acid Analysis Kit by LC MS breviatio ternate Abbreviations Alanine ALA A Arginine ARG R Asparagine ASN N Aspartic Acid ASP D Cystine C C Cys Glutamine GLN Q Glutamic Acid GLU E Histidine HIS H Hydroxylysine 2 isomers HLY HLYS OHLys 4 Hydroxyproline HYP OHPro Isoleucine ILE Leucine LEU L Lysine LYS K Methionine MET M Phenylalanine PHE F Proline PRO Serine SER 5 Threonine THR T Tyrosine TYR Y Tryptophan TRP Ww Valine VAL TRP is completely lost during acid hydrolysis use alternative hydrolysis procedure to analyze for TRP ASN and GLN are quantitatively converted to ASP and GLU during acid hydrolysis E
25. on Medium yet The freshly prepared Eluting Medium vial may be placed in one of the empty slots in the reagent tray 1 For each sample line up one glass sample preparation vial in the vial rack Figure 2 Be aware of some variability in vial opening and sorbent tip dimensions which may prevent the tip from reaching to the bottom of the sample preparation vial Note Droplets of solvent in SPE tip or spilled sorbent particles will not affect the precision of the assay in any way 2 Add the sample as follows For vapor phase hydrolysates Dry down any remaining acid in sample vial using a speed vac evaporator Pipette 100yL of Reagent 1 into sample vial to re dissolve amino acids Section 3 4 2 For liquid phase hydrolysates pipette 100uL or less of the hydrolysate sample and 200uL of Reagent 2 into a vial keep the ratio of hydrolysate reagent 2 1 2 and mix briefly The mixture should have a pH gt 1 5 but lt 5 0 Check the pH of one sample with pH paper all other samples prepared by the same procedure should have a similar pH Pipette 251 of mix and 100uL Reagent 1 into each sample preparation vial Note In either case for quantitative analysis calculate the multiplication factor by taking into account the amount of sample and volumes of HCI Reagent 2 or water used prior to SPE sample cleanup Note Amino acid standard mixtures come with the correct pH No pH adjustment is needed as described above Just add 100uL Rea
26. ot touching the inner wall of the sample vial with the tip of the Microdispenser The piston will ensure proper transfer of liquids into the vial without the need of touching the vial wall Use the same Microdispenser with both Reagents 4 and 5 There is no need to change Microdispenser tips between uses or to wash the dispenser between uses of Reagent 4 and 5 1 k Warning Do not use regular pipettes and tips with Reagents 4 and 5 as they will contaminate the sample Dedicate the Microdispenser to Reagents 4 and 5 ONLY Note for all subsequent sample preparation steps use a vortex mixer set in the touch pulse mode to about 80 of max speed for any mixing operations 12 Emulsify the liquid in the vial by repeatedly vortexing for about 5 8 seconds During vortexing hold the sample vial firmly between fingers and keep it straight as you push it onto the vortex plate Do not let the vial wobble otherwise liquid may come out of the vial Allow reactions to proceed 1 minute or more Note a longer reaction time than 1 minute at step 12 and 13 or later at step 14 does not affect results 13 Re emulsify the liquids in the vial by vortexing again for about 5 seconds Allow the reaction to proceed for one additional minute or more 14 Transfer with the Microdispenser 100uL Reagent 5 2 x 50uL for convenience and mix for about 5 seconds Let the reaction proceed for one more minute EZ faast User s Manual
27. pense 200uL aliquots of standard and Reagent 1 into each of two sample vials Perform the SPE and derivatization steps to each vial as described by the EZ faast procedure section 3 4 Transfer the organic layers from the two sample vials into one autosampler vial and evaporate to near dryness with a nitrogen stream Reconstitute 2001 of 1 2 mobile phase A B mixture and use for tuning the mass spectrometer Best results can be achieved by tuning the mass spectrometer for all analytes of interest in your particular assay Triple quadrupole mass analyzers allow tuning for a large number of ions if dwell times are chosen judiciously Nevertheless some mass spectrometers will not allow for concomitant tuning for a large number of ions as required for amino acid profiling This impediment can be easily overcome by creating time segments periods in the run file where a selected group of ions are analyzed within each segment This use of segments allows for optimal tuning for a large number of desired amino acids It is recommended that ion trap mass analyzers be tuned at least for the analytes suggested in the table below A suggested breakdown of the MS analysis into three segments looks as follows Time Suggested Tune AA AA in Range AA at End of Range 0 4 8 min SER and THR R S G T THR 4 8 9 3 min HLY and ASP A M P K D H V E W GLU or TRP 9 3 14 min LEU and C C LEI C C Y TYR A suggested breakdown of the MS analysis into six se
28. stamine HA 5 7 1111 284 198 138 p Aminobutyric acid BABA 5 8 103 1 232 130 172 88 Sarcosine SAR 5 8 89 1 218 88 158 116 10 B Aminoisobutyric acid 61 103 1 232 172 130 a Aminobutyric acid ABA 6 5 103 1 232 172 130 144 1 2 4 Diaminobutyric acid DABA 6 7 118 1 333 273 Ornithine ORN 0 6 9 132 1 347 287 156 227 1 Carnosine 6 9 226 2 441 381 284 353 Lysine alanine dipeptide LYS ALA 6 9 217 2 432 317 301 170 Methionine d3 Met d3 69 152 2 281 193 221 142 NA Methionine MET M 7 149 2 278 190 218 142 1 Proline PRO 7 3 115 1 244 156 114 184 1 S Pyridylethyl cysteine 7 5 226 3 355 106 Lysine LYS K 77 146 1 361 301 170 0 1 Aspartic acid ASP D 7 8 133 1 304 216 130 244 1 Histidine HIS H 7 8 155 1 370 196 110 284 1 EZ faast User s Manual Table 2 continued 133 2 Thiaproline TPR 7 9 174 88 202 10 Seleno methionine Se MET 8 196 1 326 238 Homoserine HSER 8 1 119 1 230 188 Valine VAL 8 2 117 1 246 158 116 186 1 Aspartame 8 3 2943 423 363 391 Glutamic acid GLU 8 3 1471 318 172 258 230 1 Tryptophan TRP 8 5 204 2 333 245 273 230 2 Carboxymethyl cysteine 8 7 179 2 350 290 Ethionine ETH 8 8 1632 292 204 y Glutamyl e lysine GLU LYS 8 8 2753 532 472 412 dipeptide a Aminoadipic acid AAA 95 161 2 332 244 185 272 Leucine LEU L 10 131 2 260 172 2 Phenyl
29. th Rubber PTFE septa AH0 4610 Silicone PTFE septa AH0 4613 PTFE Silicone PTFE septa 4616 Amber wide mouth vial cap and septa kit pack with Rubber PTFE septa AH0 4619 Silicone PTFE septa 4622 Clear wide mouth vial cap with pre slit septa Silicone PTFE septa 7507 Unit 1000 pk 1000 pk 1000 pk 1000 pk 1000 pk 1000 pk Detach Here 3882_L6 EZ faast User s Manual EZ faast Amino Acid Analysis of Protein Hydrolysates by LC MS Quick GuipE Summary of Procedure 1 For each sample line up one glass sample preparation vial in the vial rack 2 Vapor phase hydrolysate Dry down any remaining acid in sample vial using a speed vac evaporator Pipette 100 of Reagent 1 into sample vial to re dissolve amino acids Section 3 4 2 Liquid phase hydrolysate Pipette 100uL sample hydrolysate and 200 Reagent 2 into a glass vial and mix briefly If pH gt 1 5 pipette 25uL of mix and 100yL Reagent 1 into each sample preparation vial Section 3 4 2 3 Attach a sorbent tip to 1 5mL syringe pass the solution in the sample preparation vial through the sorbent tip by slowly pulling back the syringe piston Pipette 200pL water into the sample preparation vial Slowly pass the solution through the sorbent tip and into the syringe barrel Detach the sorbent tip and discard the liquid accumulated in the syringe Pipette 200 Eluting Medium prepared fresh each day sect
30. uted sample is ready for LC MS analysis LC MS Analysis LC Mobile phase 10mM Ammonium formate in water 10mM Ammonium formate in methanol Gradient 0 00min 68 B 13 00 83 B 13 01 68 B 17 00 68 B Re equilibrate column for 4 to 6 min before next injection depending on HPLC system used Flow rate 0 50mL min for 3 0mm ID column 0 25mL min for 2 0mm ID column Column temperature 35 C Injection volume MS Either ESI or may be used Mode Positive lon Scan range 100 600 m z ESI ion source temperature 365 C Bruker 425 C AB API 3000 APCI ionization chamber temperature 450 C EZ faast Kit Each kit includes a EZ faast AAA LC column or ZB AAA GC column and liners sample prep and derivatization reagents sample prep vials AA standard mixtures SPE sorbent tips vial rack autosampler vials with inserts come with MS kits Microdispenser for Reagents 4 and 5 and demo video Description GC FID Free Physiological Amino Acid Analysis Kit GC MS Free Physiological Amino Acid Analysis Kit GC FID Protein Hydrolysate Kit GC MS Protein Hydrolysate Kit LC MS Free Physiological Amino Acid Analysis Kit with 250 x 2 0mm column LC MS Free Physiological Amino Acid Analysis Kit with 250 x 3 0mm column LC MS Protein Hydrolysate Kit with 250 x 2 0mm column LC MS Protein Hydrolysate Kit with 250 x 3 0mm column GC Free Physiological Amino Acid Standards SD1 SD2 amp SD3 2mL vial x 2
31. vel samples due to background protein contamination Finally both TRP and CYS are completely destroyed by acid hydrolysis and must be analyzed by alternate methods see below The above listed limitations are based on hydrolysis chemistry and are not related at all to the EZ faast process EZ faast User s Manual 3 3 5 Alternate Methods and References For TRP analysis the use of either 4N Methane Sulfonic Acid Dodecanethiol HCl or Thiogly colic acid has been shown to generate some useful results for TRP however yields tend to be low for all of these methods For CYS analysis reduction and alkylation to generate either carboxymethyl cysteine or pyridylethyl cysteine are the preferred methods for detection for EZ faast procedure cysteic acid cannot be detected by EZ faast Procedures for useful hydrolysis methods can by found in the following references e Stein and Moore Methods in Enzymology 6 pp 819 831 1963 e Tarr in Microcharacterization of Proteins Shively ed Humana Press 1986 Miedel et al J Biochem Biophys Methods 18 pp 37 52 1989 e Strydom et al in Techniques in Protein Chemistry IV Angeletti ed 1993 e Jones et al J Liquid Chromatography 4 pp 454 486 1981 For additional information contact your Phenomenex Technical Representative 3 4 Preparation of Protein Hydrolysates for Liquid Chromatographic Analysis Please first refer to section 3 2 if you have not prepared fresh Eluti
32. wn any remaining acid in sample vial using a speed vac evaporator Pipette 100yL of Reagent 1 into sample vial to re dissolve amino acids Section 3 4 2 9 Perform EZ faast procedure as per manual 3 3 3 Liquid Phase Hydrolysis The following is a sample method for liquid phase hydrolysis as a reference other methods may work better for your application Transfer 5 25 nanomoles of protein into a glass test tube 10x150mm Lyophilize sample in a vacuum concentrator Add 100 uL of 6N containing 4 Thioglycolic acid to tube Purge air from tube with vacuum and flame seal tube Hydrolyze in oven at 110 C for 22 hours Cool tube break seal and perform EZ faast procedure as per manual n won 3 3 4 Limitations of Hydrolysis Methods While 6N acid hydrolysis is the most common procedure there are several limitations to this method ASN and GLN are deamidated to ASP and GLU and thus are quantitated as a mixture Peptide bonds of hydrophobic amino acids VAL ILE LEU may be difficult to break and require additional hydrolysis time up to 72 hours Residual oxygen in the hydrolysis vessel can increase the thermal breakdown of hydroxyl and sulfur containing amino acids typical recoveries for SER THR HYP and TYR range between 50 90 MET ranges from 25 75 Reducing hydrolysis time improves recoveries but reduces other amino acid yields see above GLY yields tend to exceed 100 especially for low le

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