2Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standardsSelecting standard peptides and building methodsPractical notes and suggestions.
3MRM/SRM/Targeted proteomics MRM: Multiple Reaction MonitoringSRM: Selective/Selected Reaction MonitoringSpecifically monitoring or ‘targeting’ one or more peptidesShotgun/untargeted proteomics: Coomassie stained gelTargeted proteomics:Western blot
4MRM/SRM/Targeted proteomics MRM: Multiple Reaction MonitoringSRM: Selective/Selected Reaction MonitoringSpecifically monitoring or ‘targeting’ one or more peptidesShotgun/untargeted proteomics: Coomassie stained gelTargeted proteomics:Western blot-Indiscriminately identifies most abundant proteins-No prior knowledge required for protein detection-Information obtained for a large number of proteins-Selectively targets one protein-Requires prior knowledge of protein mass/sequence-Limited number of proteins can be assays at once (<150)-Improved sensitivity!-Higher throughput!
5Peptides are targeted using a triple quadrupole mass spectrometer (QQQ) Triple quads contain 3 quadrupoles in series that are programed to selectively stabilize your ion of interest. Quadrupoles act as a mass filter.Ion SourceDetectorThe DC and RF voltages are tuned to stabilize particular m/z ranges
6SRM analysis uses 2 stages of mass filtering Q1q2Q3Ion SourceDetectorFragmentationQ1: Peptide mass is selected (parent ion)q2: peptide is fragmented via collision induced dissociationQ3: Peptide fragment is selected (fragment ion)Parent ion to fragment ion mass change is called a “transition”Usually ≥ 3 transitions are monitored for each peptide of interest
7SRM analysis uses 2 stages of mass filtering Q1q2Q3At1gSKNLTSSGDHMSDALSAIPAAVHRNLSDKLYEKRKNAALMLENIVKNLTSSGDHDKISKVIEMLIKEFAKSPQANHRSGDHDISKSGDHDISKAQYLEQIVPPVINSFSDQDSRVRYYACEALYDHDISKNLTSSGDHDISKNLNLTSSDISKThree transitions (aka 3 pieces of data identifying this peptide)NLTSSGDHDISKSGDHDISKNLTSSGDHDISKDHDISKNLTSSGDHDISKNLTSS
8Basic workflow for SRM analysis Extract proteinsDigest into peptidesChromatographic separation of peptides (C18 column)ESIElectrospray IonizationMS analysisParent ion selectionFragment ion selectionFragmen-tationQ1q2Q3Ion IntensityTime
9Peptides are quantified using stable isotope labeled peptide standards NLTSSGDHDISKEndogenous:Standard:NLTSSGDHDIS[K+08]Q1 mass637.67641.67Q3 fragmenty7Q3 mass779.38771.38Peptide APeptide BSingle transitionIntensitym/zEndogenousPeptide StandardEndogenousExtracted ion chromatogram (XIC)IntensityTimeStandard
10Peptides are quantified using stable isotope labeled peptide standards NLTSSGDHDISKEndogenous:Standard:NLTSSGDHDIS[K+08]Q1 mass637.67641.67Q3 fragmenty7Q3 mass779.38771.38Peptide APeptide BSingle transitionIntensitym/zEndogenousPeptide StandardOverlay: Std & Endog.Extracted ion chromatogram (XIC)IntensityTime
11Peptides can be multiplexed in a single targeted MS run Standard peptidesStandards peptidesEndogenous peptidesEndogenous peptides
12Peptide standards are spiked in during sample processing Extract proteinsDigest into peptidesChromatographic separation of peptides (C18 column)**ESISingle transitionElectrospray IonizationMS analysisIntensitym/zEndogenousPeptide Std.Parent ion selectionFragment ion selectionFragmen-tationQ1q2Q3IntensityTime
13Quantitation is achieved by measuring area under XIC curve signal intensity normalized to peptide standardEndog AreaStd. Area=
14Awesome freeware exists for analyzing SRM data MacCoss Labhttps://skyline.gs.washington.edu/Vendor specific software also exists:MultiQuant from ABSciex
15How to select peptides for SRM analysis ConsiderationsFeasibility of chemical synthesis-Peptide length(≤ 20 A.A., or ≤ 24 A.A.)-PTMs?Physiochemical properties-Hydrophobicity-Chemically modified residues(Met, Cys)Biological considerations-Is the peptide unique to 1 protein-Likelihood of trypsin misscleavagePRESENCE OF EMERPICAL MS DATA!-Has your protein been detected by MS?-Software for predicting proteotypicbehavior” of peptides is“Not so good”-Dr. MacCossGood performingPoor performingNumber of peptidesHydrophobicity bins (SSR Calc)Picotti et. al Nature Vol 494, pp
16Examples of endogenous peptide detection success rate Sussman Lab data:-Lab mate working with rat blood proteins:In silico selection ~20%Empirical data ~80%-Targeting specific Arabidopsis protein:11 tryptic peptides selected from in silico prediction, 2 endogenous peptides detected after SCX fractionation AND extended LC gradient.In silico selection ~18%-Arabidopsis phosphopeptides:65 peptides selected from discovery shotgun proteomics data, 61 endogenous peptides detected.Empirical data ~93%NOTE: Isobaric tags may influence peptide behavior. Keep this in mind when viewing discovery data from iTRAQ or TMT experiments. In general, good quality MS1 spectra is a good indicator of SRM peptide performance.Success rate for peptide detection depending on selection sourceHuttenhain et al. Sci Transl Med 11 July 2012: Vol. 4, Issue 142, p. 142ra94
17Commercial options for peptide synthesis Pros ConsGuaranteed 7-day turn around Length restriction (~20 amino acids)Cheap (around $60/peptide) Minimum order requirement (24)More PTMs available Expensive ($200-$300/peptide)No minimum order size Slow production (months)>95% pureSigma-AldrichPEPscreen Peptide librariesAQUA peptidesThermo ScientificPEPotecPros ConsCheap! (around $40/peptide)Minimum order requirement (4)Peptides arrive resuspendedNote: all prices are for heavy labeled peptides and are approximate
18Developing SRM methods What you need to know-Peptide parent mass and charge state-Fragment peptide masses and charge states-Highly recommend building SRM methods by first starting with peptide standardsResourcesMacCoss Labhttps://skyline.gs.washington.edu/
19Developing SRM methods Step 0: Successfully resolubilize lyophilized peptide standards. Recommend stepwise resuspension.Step 1: Determine strongest transitions for each peptide (start with 5/peptide; method can be trimmed down to 3/peptide later on). If your instrument has an ion trap, this process is easier.Step 2: Optimize collision energy (CE). This must be performed for every single transitions.Step 3: Determine retention time of peptide. Using scheduled SRM methods significantly improves multiplexing capability.Step 4: Look for endogenous peptides. Determine necessary pre-fractionation steps.
20Why I like targeted MS: improved peptide detection Untargeted discovery dataDetection overlap between samplesDetection overlap between injection replicatesInj1Inj2Offline SCX fraction + 4 hour LC-MS runs185306460No SCX fraction, 90min LC-MS runsComparison between MS methodsPICC pS124PEN3 pS40UnfractionatedSCX fractionStandard peptideEndogenous peptideTargeted SRM QuantitationUntargeted QuantitationIntensityTimeIntensity, cps
21Log2 (treated/control) 3 fold +1.2 fold +/-3 fold -Reliable peptide detection means proteins can be reproducibly analyzed across many different samplesColdFCJAFlg22H2O2ABANaClMann.KClAT5G56980 pS61MSL9 pS124EIF4A1 pT145JAZ12 pS97AHA1 pT948AHA2 pT947CPK5 pS552*ERD14 pS59YAK1 pY284AHA3 pT882AHA3 pT948CAX4 pS38PIP3B pS274AHA4 pT959PIP2F pS283AMT1 pS488AT5G53420 pS204NIA1 pS537NPC4 pT158DaySleeper pS155TRP1 pS214PP2C-g pS347RPS6 pS240WDL1 pS6ZAC pS155AREB3 pS43ABF2 pS86*HSFB2B pS222SnRK2.2 pS177SnRK2.3 pS176SnRK2.6.1 pS175CPK9 pS78PEN3 pS40ADH1 pS229CPK9 pT37PEPC1 pS11AHA2 pS899Remorin pT58SIP1 pS11PICC pS124Ox-reductase pS29FAC1 pS203PIP2F pS286PLC2 pS280GC5 pS793V-ATPase pS241SAY1 pS313COP related pS24MAP4Kα1 pS478VCS pS692bZIP30 pS176MyoB1 pS825PB1domain pS218RAF18 pS671TUA3 pT349*TUA4 pT349*SnRK2.4 pS158*Vac14 pS624Heat map of 5 min phosphorylation response of 60 peptides under 9 treatment conditionsABAresponsiveblockOsmotic-specificblockStecker et al. Plant Physiology (2014):
24Practical sample handling comments Targeted ProteomicsThree biological replicates per treatmentProcess all samples and controls in the SAME batch!-Extract proteins on the same day-Spike standards on the same day from the same aliquotIt is difficult to correct for differential sample handling before standard peptides are spiked in!Homogenization, protein extractionSpike in isotopically labeled peptide standards, trypsin digest,TiO2 phosphopeptide enrichment90 min LC-MS analysis using Triple Quadrupole (QQQ)Parent ion selectionFragment ion selectionFragmentationQ1Q2Q3Quantification of endogenous/standard extracted ion chromatogramsStandardm/zIntensityEndogenousTimeIntensity
25Useful references“A complete mass-spectrometric map of the yeast proteome applied to quantitative trait analysis.”Paola Picotti, (lots of authors) Reudi Aebersold (2013) Nature“Selected reaction monitoring–based proteomics: workflows, potential, pitfallsand future directions.” Paola Picotti & Ruedi Aebersold (2012) Nature Methods“Selected reaction monitoring for quantitative proteomics: a tutorial.” Vinzenz Lange, Paola Picotti, Bruno Domon and Ruedi Aebersold (2008) Molecular Systems Biology 4:222Arabidopsis SRM data from our labStecker KE et al. "Phosphoproteomic Analyses Reveal Early Signaling Events in the Osmotic Stress Response." Plant Physiology (2014):Su SH et al. "Deletion of a tandem gene family in Arabidopsis: increased MEKK2 abundance triggers autoimmunity when the MEKK1-MKK1/2-MPK4 signaling cascade is disrupted." The Plant Cell Online 25.5 (2013):