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Targeted Proteomics

Targeted Proteomics . Kelly Stecker Sussman Lab. Outline. What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides and building methods Practical notes and suggestions. MRM/SRM/Targeted proteomics. MRM: Multiple Reaction Monitoring

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Targeted Proteomics

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  1. Targeted Proteomics Kelly Stecker Sussman Lab

  2. Outline • What is MRM/SRM/Targeted mass spec • Quantification using peptide standards • Selecting standard peptides and building methods • Practical notes and suggestions.

  3. MRM/SRM/Targeted proteomics MRM: Multiple Reaction Monitoring SRM: Selective/Selected Reaction Monitoring Specifically monitoring or ‘targeting’ one or more peptides Targeted proteomics: Western blot Shotgun/untargeted proteomics: Coomassie stained gel 1 2 3

  4. MRM/SRM/Targeted proteomics MRM: Multiple Reaction Monitoring SRM: Selective/Selected Reaction Monitoring Specifically monitoring or ‘targeting’ one or more peptides Targeted proteomics: Western blot Shotgun/untargeted proteomics: Coomassie stained gel 1 2 3 -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!

  5. Peptides 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 Source Detector The DC and RF voltages are tuned to stabilize particular m/z ranges www.waters.com

  6. SRM analysis uses 2 stages of mass filtering Q1 q2 Q3 Ion Source Detector Fragmentation Q1: Peptide mass is selected (parent ion) q2: peptide is fragmented via collision induced dissociation Q3: 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

  7. SRM analysis uses 2 stages of mass filtering q2 Q3 Q1 At1g01690.1 SK NLTSSGDH MSDALSAIPAAVHRNLSDKLYEKRKNAALMLENIVKNLTSSGDHDKISKVIEMLIKEFAKSPQANHR SGDHDISK SGDHDISK DHDISK AQYLEQ IVPPVINSFSDQDSRVRYYACEALY NL NLTSSGDHDISK NLTSS DISK Three transitions (aka 3 pieces of data identifying this peptide) NLTSSGDHDISK SGDHDISK NLTSSGDHDISK DHDISK NLTSSGDHDISK NLTSS

  8. Basic workflow for SRM analysis Extract proteins Chromatographic separation of peptides (C18 column) Digest into peptides ESI Electrospray Ionization MS analysis Q1 q2 Q3 Ion Intensity Parent ion selection Fragmen-tation Fragment ion selection Time

  9. Peptides are quantified using stable isotope labeled peptide standards Q1 mass Q3 fragment Q3 mass Endogenous: Standard: Peptide Standard 637.67 y7 NLTSSGDHDISK 771.38 Endogenous NLTSSGDHDIS[K+08] 641.67 y7 779.38 Peptide A Peptide B Single transition Endogenous Intensity Intensity Extracted ion chromatogram (XIC) Time m/z Standard

  10. Peptides are quantified using stable isotope labeled peptide standards Q1 mass Q3 fragment Q3 mass Endogenous: Standard: Peptide Standard 637.67 y7 NLTSSGDHDISK 771.38 Endogenous NLTSSGDHDIS[K+08] 641.67 y7 779.38 Peptide A Peptide B Single transition Overlay: Std & Endog. Intensity Intensity Extracted ion chromatogram (XIC) Time m/z

  11. Peptides can be multiplexed in a single targeted MS run Standard peptides Standards peptides Endogenous peptides Endogenous peptides

  12. * * * * * * * * * * * * * * * * Peptide standards are spiked in during sample processing Extract proteins Chromatographic separation of peptides (C18 column) Digest into peptides Peptide Std. ESI Endogenous Single transition Electrospray Ionization MS analysis Q1 q2 Q3 Intensity Intensity Parent ion selection Fragmen-tation Fragment ion selection Time m/z

  13. Quantitation is achieved by measuring area under XIC curve signal intensity normalized to peptide standard Endog Area Std. Area =

  14. Awesome freeware exists for analyzing SRM data MacCoss Lab https://skyline.gs.washington.edu/ Vendor specific software also exists: MultiQuant from ABSciex

  15. How to select peptides for SRM analysis • Considerations • Feasibility 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 misscleavage • PRESENCE OF EMERPICAL MS DATA! • -Has your protein been detected by MS? • -Software for predicting proteotypic • behavior” of peptides is • “Not so good”-Dr. MacCoss Good performing Poor performing Number of peptides Hydrophobicity bins (SSR Calc) Picotti et. al. 2013 Nature Vol 494, pp 266-270

  16. Examples 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 source Huttenhainet al. SciTransl Med 11 July 2012: Vol. 4, Issue 142, p. 142ra94

  17. Commercial options for peptide synthesis Pros Cons Guaranteed 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% pure • Sigma-Aldrich • PEPscreen Peptide libraries • AQUA peptides • Thermo Scientific • PEPotec Pros Cons Cheap! (around $40/peptide) Minimum order requirement (4) Peptides arrive resuspended http://www.sigmaaldrich.com/life-science/custom-oligos/custom-peptides/product-highlights/pepscreen-peptides.html http://www.piercenet.com/info/pepotec-srm-custom-peptide-libraries Note: all prices are for heavy labeled peptides and are approximate

  18. Developing 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 standards Resources MacCoss Lab https://skyline.gs.washington.edu/ http://prospector.ucsf.edu/prospector/mshome.htm

  19. Developing 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.

  20. Why I like targeted MS: improved peptide detection Untargeted discovery data Detection overlap between samples Detection overlap between injection replicates 306 460 185 Offline SCX fraction + 4 hour LC-MS runs Inj1 Inj2 No SCX fraction, 90min LC-MS runs Untargeted Quantitation Targeted SRM Quantitation Unfractionated SCX fraction Standard peptide Endogenous peptide PICC pS124 PICC pS124 Intensity Intensity, cps Comparison between MS methods PEN3 pS40 PEN3 pS40 Intensity Intensity, cps Time Time

  21. Log2 (treated/control) Reliable peptide detection means proteins can be reproducibly analyzed across many different samples 3 fold + 1.2 fold +/- 3 fold - Mann. Cold FC JA Flg22 H2O2 ABA NaCl KCl AT5G56980 pS61 MSL9 pS124 EIF4A1 pT145 JAZ12 pS97 AHA1 pT948 AHA2 pT947 CPK5 pS552* ERD14 pS59 YAK1 pY284 AHA3 pT882 AHA3 pT948 CAX4 pS38 PIP3B pS274 AHA4 pT959 PIP2F pS283 AMT1 pS488 AT5G53420 pS204 NIA1 pS537 NPC4 pT158 DaySleeper pS155 TRP1 pS214 PP2C-g pS347 RPS6 pS240 WDL1 pS6 ZAC pS155 AREB3 pS43 ABF2 pS86* HSFB2B pS222 SnRK2.2 pS177 SnRK2.3 pS176 SnRK2.6.1 pS175 CPK9 pS78 PEN3 pS40 ADH1 pS229 CPK9 pT37 PEPC1 pS11 AHA2 pS899 Remorin pT58 SIP1 pS11 PICC pS124 Ox-reductase pS29 FAC1 pS203 PIP2F pS286 PLC2 pS280 GC5 pS793 V-ATPase pS241 SAY1 pS313 COP related pS24 MAP4Kα1 pS478 VCS pS692 bZIP30 pS176 MyoB1 pS825 PB1domain pS218 RAF18 pS671 TUA3 pT349* TUA4 pT349* SnRK2.4 pS158* Vac14 pS624 Heat map of 5 min phosphorylation response of 60 peptides under 9 treatment conditions ABA responsive block Osmotic-specific block Stecker et al. Plant Physiology 165.3 (2014): 1171-1187.

  22. Log2 (treated/control) Reliable peptide detection means proteins can be reproducibly analyzed across many different samples 3 fold + 1.2 fold +/- 3 fold - Mann. Cold FC JA Flg22 H2O2 ABA NaCl KCl AT5G56980 pS61 MSL9 pS124 EIF4A1 pT145 JAZ12 pS97 AHA1 pT948 AHA2 pT947 CPK5 pS552* ERD14 pS59 YAK1 pY284 AHA3 pT882 AHA3 pT948 CAX4 pS38 PIP3B pS274 AHA4 pT959 PIP2F pS283 AMT1 pS488 AT5G53420 pS204 NIA1 pS537 NPC4 pT158 DaySleeper pS155 TRP1 pS214 PP2C-g pS347 RPS6 pS240 WDL1 pS6 ZAC pS155 AREB3 pS43 ABF2 pS86* HSFB2B pS222 SnRK2.2 pS177 SnRK2.3 pS176 SnRK2.6.1 pS175 CPK9 pS78 PEN3 pS40 ADH1 pS229 CPK9 pT37 PEPC1 pS11 AHA2 pS899 Remorin pT58 SIP1 pS11 PICC pS124 Ox-reductase pS29 FAC1 pS203 PIP2F pS286 PLC2 pS280 GC5 pS793 V-ATPase pS241 SAY1 pS313 COP related pS24 MAP4Kα1 pS478 VCS pS692 bZIP30 pS176 MyoB1 pS825 PB1domain pS218 RAF18 pS671 TUA3 pT349* TUA4 pT349* SnRK2.4 pS158* Vac14 pS624 20%CV Median Standard Deviation Average CV=

  23. Log2 (treated/control) Reliable peptide detection means proteins can be reproducibly analyzed across many different samples 3 fold + 1.2 fold +/- 3 fold - Mann. Cold FC JA Flg22 H2O2 ABA NaCl KCl AT5G56980 pS61 MSL9 pS124 EIF4A1 pT145 JAZ12 pS97 AHA1 pT948 AHA2 pT947 CPK5 pS552* ERD14 pS59 YAK1 pY284 AHA3 pT882 AHA3 pT948 CAX4 pS38 PIP3B pS274 AHA4 pT959 PIP2F pS283 AMT1 pS488 AT5G53420 pS204 NIA1 pS537 NPC4 pT158 DaySleeper pS155 TRP1 pS214 PP2C-g pS347 RPS6 pS240 WDL1 pS6 ZAC pS155 AREB3 pS43 ABF2 pS86* HSFB2B pS222 SnRK2.2 pS177 SnRK2.3 pS176 SnRK2.6.1 pS175 CPK9 pS78 PEN3 pS40 ADH1 pS229 CPK9 pT37 PEPC1 pS11 AHA2 pS899 Remorin pT58 SIP1 pS11 PICC pS124 Ox-reductase pS29 FAC1 pS203 PIP2F pS286 PLC2 pS280 GC5 pS793 V-ATPase pS241 SAY1 pS313 COP related pS24 MAP4Kα1 pS478 VCS pS692 bZIP30 pS176 MyoB1 pS825 PB1domain pS218 RAF18 pS671 TUA3 pT349* TUA4 pT349* SnRK2.4 pS158* Vac14 pS624 P-value +/- 1.25 fold change +/- 1.5 fold change 0.05 Students T-Test: 3 control samples, 3 treated samples

  24. Practical sample handling comments Targeted Proteomics Three biological replicates per treatment Process all samples and controls in the SAME batch! -Extract proteins on the same day -Spike standards on the same day from the same aliquot It is difficult to correct for differential sample handling before standard peptides are spiked in! Homogenization, protein extraction Spike in isotopically labeled peptide standards, trypsin digest, TiO2 phosphopeptide enrichment 90 min LC-MS analysis using Triple Quadrupole (QQQ) Quantification of endogenous/standard extracted ion chromatograms Intensity Intensity Q1 Q2 Q3 Standard Time m/z Endogenous Parent ion selection Fragmentation Fragment ion selection

  25. Useful references • “A complete mass-spectrometric map of the yeast proteome applied to quantitative trait analysis.” • Paola Picotti, (lots of authors) ReudiAebersold (2013) Nature • “Selected reaction monitoring–based proteomics: workflows, potential, pitfalls • and future directions.” Paola Picotti & RuediAebersold (2012) Nature Methods • “Selected reaction monitoring for quantitative proteomics: a tutorial.” Vinzenz Lange, Paola Picotti, Bruno Domon and RuediAebersold (2008) Molecular Systems Biology 4:222 • Arabidopsis SRM data from our lab • Stecker KE et al. "Phosphoproteomic Analyses Reveal Early Signaling Events in the Osmotic Stress Response." Plant Physiology 165.3 (2014): 1171-1187. • 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): 1895-1910.

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