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2ProceduresPreparationPreparationTo prepare trypsinCAUTIONDo not freeze and thaw the trypsin more than five times after the lyophilized powder isresuspended, or you can have loss or reduction of activity.1 Dissolve the lyophilized proteomics grade trypsin in 100 µL of 50 mM aceticacid, to a final concentration of 1 µg/µL.2 Mix by gently pipetting up and down.3 Keep the trypsin solution on ice and transfer the solution into 10 aliquots of10 µL each.Store the aliquots at - 20 C for up to one month or at - 80 C for long termstorage.During use1 Thaw the reconstituted proteomics grade trypsin at room temperature.2 Place immediately on ice.3 Remove the amount of enzyme required for the experiment and thenrefreeze the unused portion. Do not exceed five freeze- thaw cycles.To prepare reagents and supplies Wash non- stick (low protein binding) microcentrifuge tubes with 100%ethanol. Allow the tubes to dry completely. Use only ultrapure H2O ( 18 MΩ) for reagent preparation.14Proteomics Grade Trypsin Protocol

ProceduresTo prepare gel for In-Gel Digestion protocol2To prepare gel for In-Gel Digestion protocolEach in- gel digestion reaction is designed for the preparation of proteolyticpeptides from a protein band or spot after 1- D or 2- D electrophoreticseparation of protein samples.1 Do SDS- PAGE using polyacrylamide gels that are 1 to 1.5 mm thick.2 Stain the gels with Coomassie blue stain or silver stain. (For silver stainedgels, you need to do a mass spectrometry- compatible silver destainingprocedure before you do the gel core destaining protocol.)*Best results are achieved when the amount of protein in the excised band is 1 pmol. This approximately corresponds to the sensitivity of Coomassiestain- based detection.3 Excise a gel core ( 1 mm 4 mm) that contains the protein spot or band ofinterest from the polyacrylamide gel.4 Carefully cut the gel using a clean scalpel or razor blade. Cut as close aspossible to the stained protein band or spot. To facilitate gel core excision,place the gel on a glass plate in a puddle of H2O before you cut the gel.5 Clean the blade and the glass plate with ethanol before use.6 Cut the gel core into 1 mm3 pieces and put the gel pieces in non- stick1.5- mL microcentrifuge tubes (see “To prepare reagents andsupplies” on page 14). Use a clean spatula or the edge of a razor blade.7 Spin the tube in a microcentrifuge at 14K rpm for 10 seconds to collect thegel pieces at the bottom of the tube.* Shevchenko, A., Wilm, M., Vorm, O. and Mann, M. (1996) Anal Chem. 68(5):850-8.Proteomics Grade Trypsin Protocol15

2ProceduresProtocol 1: In-Gel DigestionProtocol 1: In-Gel DigestionStep 1. Gel core destaining, protein reduction and alkylation1 Prepare gel cores. See “To prepare gel for In- Gel Digestion protocol” onpage 15.Volumes of reagents in this step are appropriate for gel cores of3 1 mm 4 mm 1 mm. If the gel core is significantly larger than 4 mm ,increase the volumes of reagents accordingly.2 Destain silver- stained gels with a mass spectrometry- compatible silverdestaining protocol.3 Add 200 μL of wash solution (50% CH3CN, 50 mM NH4HCO3) to the sampletube.4 Mix the sample on a vortex mixer continuously for 10 minutes at roomtemperature.5 Remove and discard the liquid using a pipette. Repeat two more times for atotal of three washes.6 Add 200 µL of acetonitrile (CH3CN) to the gel core.7 Mix on a vortex briefly, then incubate the tube at room temperature for 5 minutes.8 Remove and discard the liquid. The gel pieces will appear shrunken and canappear opaque at the end of this treatment.9 Remove the residual acetonitrile from the destained gel core sample with aheated vacuum centrifugal concentrator for 5 minutes.10 Prepare a solution of 10 mM DTT in 50 mM NH4HCO3.NOTEPrepare fresh iodoacetamide and DTT solutions just before use.11 To reduce the protein sample, add 100 µL of the freshly prepared DTTsolution to the dried gel core and incubate the tube at 55 C for 1 hour.Remove and discard the liquid.12 Prepare a solution of 55 mM iodoacetamide in 50 mM NH4HCO3.13 To alkylate the protein sample, add 100 µL of the freshly preparediodoacetamide solution to the sample tube and mix on a vortex.16Proteomics Grade Trypsin Protocol

ProceduresStep 1. Gel core destaining, protein reduction and alkylation214 Incubate the tube in the dark at room temperature for 45 minutes. Removeand discard the liquid.15 Add 200 µL of wash solution (50% CH3CN, 50 mM NH4HCO3) to the sampletube. Mix the tube on a vortex continuously for 10 minutes at roomtemperature.16 Remove and discard the liquid. Repeat two additional times for a total ofthree washes.17 Add 200 µL of acetonitrile (CH3CN) to the gel core.18 Mix briefly on a vortex mixer, then incubate the tube at room temperaturefor 5 minutes.19 Remove and discard the liquid.20 Remove the residual acetonitrile from the destained gel core sample with aheated vacuum centrifugal concentrator for 5 minutes.Proteomics Grade Trypsin Protocol17

2ProceduresStep 2. In-gel digestion with trypsin and peptide extractionStep 2. In-gel digestion with trypsin and peptide extractionYou have two options to do this step.In the first option (Conventional Digestion Protocol), the in- gel digestion isdone overnight at 37 C. In the second option (Accelerated Digestion Protocol),the in- gel digestion is done for 30 minutes at 55 C. The first option producesmore complete protein digestion. Use it when you need to obtain maximumsequence coverage or when you are analyzing proteins that are refractory todigestion by trypsin.Option 1: Conventional Digestion ProtocolThis protocol requires approximately 12 hours (digestion is done overnight).1 Reconstitute the proteomics grade trypsin. See “To prepare trypsin” onpage 14.2 Dilute an appropriate volume of the 1 µg/µL trypsin to 20 ng/µL in 50 mMNH4HCO3.NOTEStore the unused portion of concentrated trypsin at -20 C for up to one month or at -80 Cfor long-term storage. Divide into small enough aliquots to avoid multiple freeze-thawcycles.3 Add 15 µL of the 20 ng/µL trypsin solution to the dried gel core.4 Incubate the sample for 1 hour at 30 C to allow gel rehydration.5 Add sufficient digestion buffer (50 mM NH4HCO3/10% CH3CN) to thesample tube to completely cover the gel pieces. Make sure that the tube iscapped tightly to prevent evaporation.6 Incubate the tube at 37 C overnight.7 Add 50 µL of ultrapure ( 18 MΩ) H2O to the in- gel digestion mixture.8 Mix the tube on a vortex mixer continuously for 10 minutes at roomtemperature.9 Remove and keep the liquid in a fresh, non- stick microcentrifuge tube.10 Add 50 µL of 50% CH3CN/5% (v/v) formic acid to the gel pieces.11 Incubate the tube for 60 minutes at room temperature. Mix frequently on avortex mixer.18Proteomics Grade Trypsin Protocol

ProceduresStep 2. In-gel digestion with trypsin and peptide extraction212 Spin the tube briefly in a microcentrifuge and then collect the liquid with apipet. Add the liquid to the liquid collected in step 9.13 Reduce the volume of the pooled liquid from the previous step. Use acentrifugal concentrator (at room temperature), until the appropriatevolume (typically 10 to 20 µL) is reached.14 If you want to desalt or concentrate the sample before mass spectrometry,use a reverse- phase C- 18 cleanup pipette tip (Agilent p/n 5188- 5239) topurify the peptides.If online desalting will be used as part of an LC- MS method, then theextracted peptide solution may be used directly after centrifugalconcentration.Option 2: Accelerated Digestion ProtocolYou need approximately one hour to complete this protocol.1 Reconstitute the proteomics grade trypsin. See “To prepare trypsin” onpage 14.2 Dilute an appropriate volume of the 1 µg/µL trypsin to 100 ng/µL indigestion buffer containing 50 mM NH4HCO3, 5 mM CaCl2.NOTEStore the unused portion of concentrated trypsin at -20 C for up to one month or at -80 Cfor long-term storage. Divide into small enough aliquots to avoid multiple freeze-thawcycles.3 Add 3 µL of the 100 ng/µL trypsin- digestion buffer solution to the dried gelcore.4 Incubate the sample for 5 minutes at room temperature.NOTEIf you want to reduce the size of the autolytic trypsin fragment peak (m/z 842.5099) in themass spectrum, reduce the amount of trypsin to 1.5 to 2 µL.5 Add 17 µL of freshly- prepared, room temperature digestion buffer (50 mMNH4HCO3, 5 mM CaCl2) to the sample tube.6 Incubate the tube at 55 C for 30 minutes. The digestion buffer should justcover the gel pieces.To limit condensation in the sample tube, use a dry 55 C incubator insteadof a water bath.Proteomics Grade Trypsin Protocol19

2ProceduresStep 2. In-gel digestion with trypsin and peptide extraction7 Extract the peptides from the gel core: Add 15 µL of 5% (v/v) formic acid tothe tube that contains the gel pieces, enzyme and digestion buffer. Mix thetube on a vortex mixer continuously for 15 minutes at room temperature.8 Briefly spin the tube in a microcentrifuge.9 If sample desalting or concentration is desired prior to performing massspectrometry, purify the peptides using a reverse- phase C- 18 cleanuppipette tip.10 If online desalting will be used as part of an LC- MS method, then theextracted peptide solution may be used directly. In this case, spin thesample tube at 14K rpm for 1 minute and then remove a suitable volume ofthe supernatant for LC- MS analysis.20Proteomics Grade Trypsin Protocol

ProceduresProtocol 2: Protein Digestion in Solution2Protocol 2: Protein Digestion in SolutionStep 1. Protein reduction and denaturationComplete digestion requires reduction of disulfide bonds and denaturation ofthe protein. For partial digestion of native proteins, the following steps may beomitted. Ensure that the native protein sample is in a buffer with pH 7–9before proceeding to the Digestion with Trypsin (In Solution) section.1 Dissolve the protein in sample buffer that contains 50 mM Tris- HCl (pH 8),5 mM DTT, and 8 M urea to a final protein concentration of approximately1 mg/mL.2 Incubate the dissolved protein sample at 60 C for 60 minutes. After theincubation period, allow the sample to cool to room temperature.3 Add a sufficient amount of dilution buffer [50 mM NH4HCO3 (pH 7.8)] tobring the urea concentration to less than 1M.Step 2. Digestion with trypsin (in-solution)1 Add the appropriate volume of MS grade trypsin (1 µg/µL stock) such thatthe ratio of trypsin:protein in the sample is between 1:20 and 1:100 (w/w).2 Incubate the digestion reaction at 37 C for 1 hour. Digestion times willvary. Some proteins require up to 24 hours for complete digestion.3 To monitor the extent of protein digestion, remove an aliquot of the sampleand analyze with an appropriate method (e.g. gel electrophoresis orreverse- phase HPLC). If protein digestion is incomplete, return the sampleto 37 C for the appropriate amount of time to allow further digestion.4 Terminate the digestion reaction by adding formic acid to a finalconcentration of 5% (v/v). Remove a 10 to 20 µL aliquot that contains 1 to2 µg protein, and separate the protein from urea and other contaminantsusing a reverse- phase C- 18 cleanup pipette tip (Agilent p/n 5188- 5239).Proteomics Grade Trypsin Protocol21

222ProceduresStep 2. Digestion with trypsin (in-solution)Proteomics Grade Trypsin Protocol

Proteomics Grade TrypsinProtocol3TroubleshootingIf peaks are completely absent in the mass spectrum 24If too many peaks are in the mass spectrum 24If you get low-quality mass spectrum 25If you fail to determine protein identity after data mining 25If protein is identified with low confidence or low apparent sequencecoverage 25Agilent Technologies23

3TroubleshootingIf peaks are completely absent in the mass spectrumIf peaks are completely absent in the mass spectrumFailure to observe any peptide peaks may indicate that one or more protocolsteps were not completed properly. Protein losses may have occurred during storage. If eluted peptides must bestored prior to analysis, use non- stick or low- protein- binding tubes (e.g.VWR, catalog #20170- 315). Check that the correct reagents and reaction conditions (i.e. incubationtemperature and duration) were used throughout the protocol.If too many peaks are in the mass spectrumThe sample may be contaminated with keratin or other exogenous proteins. Wear gloves throughout the procedure and take care to use implements(razor blades, spatulas and glass plates) that have been freshly cleaned withethanol.24Proteomics Grade Trypsin Protocol

TroubleshootingIf you get low-quality mass spectrum3If you get low-quality mass spectrumThe quantity of protein in the gel may be too low. Repeat the 1- D or 2- D electrophoresis and load a greater amount of theprotein sample. Use a gel slice containing 1 pmol of protein (an amountthat can typically be visualized after staining with Coomassie dye). Use anarrower pH gradient for the first dimension of a 2D electrophoresisexperiment if necessary. Check that the gel slice is cut into 1 mm3 pieces before you start theprotocol. Peptide extraction is inefficient from large gel slices. Do not freeze and thaw the reconstituted trypsin multiple times. Limit thenumber of freeze- thaw cycles to five. Protein losses may have occurred during storage. If eluted peptides must bestored prior to analysis, use non- stick or low- protein- binding tubes (e.g.VWR, catalog #20170- 315).If you fail to determine protein identity after data miningThe protein that was isolated and analyzed may not be included in thedatabase being queried.If protein is identified with low confidence or low apparentsequence coverage Few proteolytic peptides were generated and available for analysis due tovery low protein abundance in the sample. See “If you get low- qualitymass spectrum” on page 25 for suggestions.Proteomics Grade Trypsin Protocol25

326TroubleshootingIf protein is identified with low confidence or low apparent sequence coverageProteomics Grade Trypsin Protocol

www.agilent.comIn This BookThis document describes howto use Proteomics GradeTrypsin to prepareproteolytic peptides for massspectrometric analysis. Agilent Technologies, Inc. 2010Version A, January 2010*204310-12*204310-12Agilent Technologies

implied warranties of merchant-ability and fitness for a particular purpose. Agilent shall not be lia-ble for errors or for incidental or consequential damages in con-nection with the furnishing, use, or performance of this document or of any information contained he