PRMs for PTM Studies with Skyline or How I learned to ditch the triple quad and love the QE Jacob D Jaffe Skyline Webinar July 2015 Outline Definitions When do PRM assays make sense Considerations for PRM method development ID: 1043292
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1. “Research-grade” Targeted Proteomics Assay Development:PRMs for PTM Studies with Skylineor,“How I learned to ditch the triple quad and love the QE”Jacob D. JaffeSkyline Webinar July 2015
2. OutlineDefinitionsWhen do PRM assays make sense?Considerations for PRM method developmentLive Demo
3. Definition of PRMPRM = MRM-HR = HR-MRM = Targeted Full Scan MS/MSClosest spiritual cousin is triple-quad based MRM/SRM, but:SRMQ3 mass analyzerm/z1m/z2m/z3PRMmass analyzerm/z rangeb1y1y4b6y5-H2Oy8y5P-H3PO4Discrete transitions, hardware selectedFull scan
4. Definition, continuedAssay is completely deterministicPrecursor m/z (list) is specifiedPossibly scheduledQuadrupole or ion trap selection/isolationFragmentation is performedAny kind is OKFull MS/MS spectrum is recordedAny analyzer: Orbitrap, TOF, scanning quad, ion trap, etc.Usually a full scan MS spectrum is also periodically recordedTwo chances to verify and quantify!
5. Common configuration: high resolution mass analyzerOrbitrap or TOFPrecursor cycle vs. Acquisition loop cyclePrecursor cycle: time it takes to loop through precursor listMay vary during methodGoverns points across peakAcquisition loop cycle: Time from full scan to full scan with intervening # of MS/MSMay affect instrument performance, full scan points across peakMS1MS21MS22MS2…MS2n
6. When do PRMs make sense?Exquisite selectivity requiredUnit (quadrupole) vs. ppm (hi-res)Post-translational modification localization is requiredGVDQ(pS)PLTPAGGK vs. GVDQSPL(pT)PAGGKRapidly convert discovery data to targeted assayStay within platformYou don’t have a triple quad!But still want the benefits of targeted proteomics
7. High resolution adds value to selectivity20xKme3SAPATGGVKprKprPHR10 KacSAPATGGVKprKprPHR10 20x20x20xABI 5500 triple quadABI 5500 triple quadQEQEm/z 551.9940 z=3m/z 551.9819 z=3
8. PTM Localization – shared ions, differential ionsDifferentially Phosphorylated PeptidesWith Same Base Sequence
9. Discovery Proteomics to PRM – Short version / Label FreeShotgun MS/MS*Database SearchTarget SelectionSpectral LibrarySkyline DocMethod/Precursor List ExportAcquire DataSkyline Quant* iRT peptides recommended
10. Discovery Proteomics to PRM – Long versionAcquire DataSkyline QuantShotgun w/ iRTDatabase SearchTarget SelectionMake Labeled PeptidesQC / Spectral LibraryiRT ModelTransition, Scheduling and Gradient Optimization in BackgroundSkyline DocExternal Data
11. Planning ahead for successStrongly consider including iRT peptides in every single sample you run in your labDiverse retention times, well spacedHigh enough levels to trigger MS/MSOr, include targeted scansOr, determine RTs with precursor quant in SkylineThis can also be very beneficial for scheduling tight windowsUse a search engine supported by Skyline spectral library importSet up your funky PTMs in advance in your documentLearn about Skyline’s secret PTM notation for import
12. Document refinementKeep a lot of transitions around initiallyYou can always get rid of them laterYou can take them from the spectral librariesIn theory: the more transitions, the more signal-to-noiseAlso in theory more sensitive than MRM, but generally not in practiceTake advantage of the raw data spectrum viewer functionalitiesHelpful for both MS and MS/MS inspectionUse that high res!Narrow your import m/z tolerancesInspect the ppm errors
13. The all important dotpdotp = dot productMetric of observed transition relative intensities in comparison with spectral library exampleBetter than a search engine score!Expect > 0.9 under most circumstancesExtremely useful in differentiating among similar analytesSpectral library quality importantGarbage in, garbage out
14. Standardization ConsiderationsLabel freeRequires high degree of system reproducibilityHard to compare samples longitudinallySynthetic peptidesHighest degree of rigorHighest cost in time, $More optimization requiredSILACIncreases complexity, chance for interferenceStandard is “prenormalized”Consider growing up a vat of standard for longitudinal performanceChemical labels? (+ standards?)
15. Data analysis considerationsBe patient, use all metrics at your disposalConsider time window import limitsBut relies on RT or other indicators in spectral library / RT modelsConsider further minimizing your document when happy with dataHi-res data, skyd files get big
16. “Research Grade” PRM ConceptA quantitative, targeted proteomics assay suitable for “everyday” useIdeally standardized with synthetic peptides (or SILAC)Rapid design cycle using discovery data/platformEnables longitudinal comparisons across days, months, yearsOutput useful for rapidly guiding biologyNOT:Obsessed with LOD/LOQSuitable for clinical deployment
17. Histones and their post-translational modificationsSource: PDB 1AOI, Luger et al. Nature (1997)NH2-A R T K Q T A R KS T G G K A P R K Q…phacmemememeAssociated with transcriptional regulatory states of genomic loci
18. Sample preparation process and standardizationAgilent Bravo LH – fully automated96-well SPE – semi-automatedDay 1Day 2Day 3