Target Analses in Parallel Reaction Monitoring Mode (PRM) Skline Webinar Januar 13, 2015 Bruno Domon, PhD Head Luxembourg Clinical Proteomics Center Invited Professor Universit of Luxembourg
INTRODUCTION TARGET QUANTITATIVE ASSAYS Domon - 2015 Skline Webinar 2
Characteristics of Quantitative Assas Sensitivit Detection of abundance components o Wide range of protein concentrations o Need for low LoD / LoQ o Wide dnamic range Scale Biological variabilit o Need to perform large studies o Throughput, i.e. robust platform o Multiplexing capabilit Selectivit Complexit of proteomic samples o Reduce sample complexit (interferences in measurements) o High resolution instruments: LC MS Domon - 2015 Skline Webinar 3
Tpes of Targeted Experiments Classical Quantitative Experiment o Precise quantification (biomarkers) o Internal standards (calibrated amount) o Limited number of analtes Sensitivit Scale Selectivit Screening Experiment o Detection of peptides in complex matrix (e.g. blood or urine samples) o Large scale (hundred of candidates) o Multiplexing capabilit Sensitivit Gallien et al., J. Mass Spectrom. 2011 Scale Selectivit Domon - 2015 Skline Webinar 4
Selected Reaction Monitoring (SRM) Triple quadrupole instrument Quantification (Traces; AUC) min Identit confirmation 513 789 827 975 1018 m/z Kim et al., Proteomics Clin. Appl. 2013 Domon - 2015 Skline Webinar 5
Targeted Proteomics SRM experiments: triple quadrupole instrument - reference method Q1 CID Q3 Fixed Fixed Limitations: o Actual number of transitions to be monitored o Low resolution mass analzers (both Q1 and Q3) > co-isolation of interferences along with the precursor ion Interference Signal observed Analte [m/z] Gallien et al., J. Mass Spectrom. 2011 Domon - 2015 Skline Webinar 6
Selectivit is an Issue.. Expected 3 4 5 6 25 27 29 31 Retention time (min) Observed "One train can hide another one! " Check twice! 3 4 5 6 25 27 29 31 Retention time (min) Domon - 2015 Skline Webinar 7
Intensit (coutns/s) Intensit (counts/s) Intensit (coutns/s) Intensit (counts/s) Intensit (coutns/s) Intensit (coutns/s) Selectivit of Measurements 5E04 5.E04 SRM NLLSVAYK 4E05 4.E05 PRM NLLSVAYK Intensit (counts/s) 5E04 4.E04 5E04 3.E04 5E04 2.E04 5E04 1.E04 3 4 5 6 Intensit (counts/s) 3E05 3.E05 2E05 2.E05 1E05 1.E05 3 4 5 6 0.E00 22 24 26 28 Elution Retention time time (min) (min) 0.E00 25 27 29 31 Retention Elution time time (min) (min) 8E03 8.E03 NLLSVAYK 8E03 8.E03 NLLSVAYK 6E03 6.E03 4E03 4.E03 3 4 5 6 6E03 6.E03 4E03 4.E03 3 4 5 6 2E03 2.E03 2E03 2.E03 0.E00 0.E00 22 24 26 28 25 27 29 31 Elution Retention time time (min) (min) Retention Elution time time (min) (min) Kim et al., Proteomics Clin. Appl. 2013 Domon - 2015 Skline Webinar 8
PARALLEL REACTION MONITORING (PRM) Domon - 2015 Skline Webinar 9
Parallel Reaction Monitoring (PRM) o Performed on a quadrupole / orbitrap instrument (high-resolution) Source MS-1 CID MS-2 Fixed m/z o Simplified experimental design o Selection of transitions, extraction of traces post-acquisition. Data Analsis time Gallien et al., J. Proteomics 2014 Domon - 2015 Skline Webinar 10
Design of a Targeted Experiment: PRM Mode Quadrupole Orbitrap / PRM mode o Simplified Method Design o Flexible Data Analsis Peptide Selection Transition Selection Optimization > Protein_1 > Protein_2 > Protein_i Peptides Pep Q1 Frag CE Time Win Fill time Area Pep_A 724 786 30 25.3 0.7 50 Pep_A 887 30 Pep_A 966 30 Pep_A 1087 30 Pep_B 863 754 35 17.5 0.7 50 Pep_B 917 35 Pep_B 1018 35 Pep_B 1132 35 Pep_N 563 797 25 24.3 0.7 50 Pep_N 899 25 Pep_N 999 25 Pep_N 1101 25 Domon - 2015 Skline Webinar 11
Parallel Reaction Monitoring Mode (PRM) 1. Isolation 2. Fragmentation 3. Transfer in OT 4. HR Analsis Domon - 2015 Skline Webinar 12
Parallel Reaction Monitoring Experiment 1 2 PRM mode (multiplexing capabilit) Q-orbitrap instrument 3 789 975 975 827 827975 535827 1018 535 1018 513 m/z 1018 m/z m/z Full MS/MS Spectra 789 Fingerprint to confirm the analte identit Ion Chrom. extraction Chrom. traces for quantification min 975 513 Kim et al., Proteomics Clin. Appl. 2013 Domon - 2015 Skline Webinar 13
Quantification Methods in PRM Sequential: Iterative analses o Sequential isolation / fragmentation events o Multiple detection scans Pep1 Pep2 Pep3 t Multiplexed: Parallel analsis o Sequential isolation and fragmentation o Intermediate storage o Single detection scan Pep1, Pep2, Pep3 Isolation (Q) Fragmentation (Coll Cell) Detection of fragments (OT) t Gallien et al., Mol. Cell. Proteomics 2012 Domon - 2014 Skline Webinar 14
Area ratio PRM Mode: Multiplexed Analsis o Sequential isolation of L/H precursors o Fragmentation and storage in HCD cell o One orbitrap detection scan Q11_0908 #8001 RT: 30.89 AV: 1 NL: 8.40E5 Q11_0908 #8001 RT: 30.89 AV: 1 F: NL: FTMS 8.40E5 p NSI Full msx ms2 642.29@hcd28.90 647.29@hcd29.13 [100.00-1340.00] F: FTMS p NSI Full msx ms2 642.29@hcd28.90 647.29@hcd29.13 [100.00-1340.00] 771.377 100 771.377 100 90 7 90 EGYYGYTGAFR (L) / 80 EGYYGYTGAFR (H) o Quantification based fragment ions o Selection of ions post-acquisition 80 70 70 60 8 0.33 0.33 60 934.440 50 L H 934.440 50 5 40 551.293 7 40 551.293 30 6 714.355 30 4 350.134 20 714.355 450.245 350.134 8 20 450.245 5 2 10 4 6 2 10 3 3 0 0 300 400 500 600 0.01 700 800 900 1000 0.01 1100 1200 300 400 500 600 700 800 900 1000 m/z 1100 1200 31 32 m/z 30 30.5 31 31.5 32 Relative Abundance 9.00 Quantification similar to SRM, but using high resolution fragment ions L Area ratio H 9.00 30 30.5 31 31.5 31 32 0.11 30 0.11 30.5 31 Retention Time 31.5 3.00 (min) 32 Retention Time (min) Retention Time (min) Amount (fm Domon - 2015 Skline Webinar 15
Fold change Fold change Fold change PRM Analses of Plasma Samples 1 0.8 0.6 0.4 0.2 SRM Data Poor correlation Peptides FIIPQIVK and LIAPVAEEEATVPNNK Surrogates: L-lactate dehdrogenase Plasma samples (AlbIgG depleted) Pilot stud: 3 controls and 3 disease 0 Control 1 Control 2 Control 3 Patient 1 Patient 2 Patient 3 LIAPVAEEEATVPNNK FIIPQIVK 1 PRM Data Excellent consistenc 1 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0 Control 1 Control 2 Control 3 Patient 1 Patient 2 Patient 3 Control 1 Control 2 Control 3 Patient 1 Patient 2 Patient 3 0Domon - 2015 Skline Webinar 16
Selectivit in HR/AM Mode /1 SRM on Triple quadrupole PRM on Q-Orbitrap Res Q3 = 0.7 XIC 700 ppm 32.5 33.5 34.5 RT (min) 38.5 39.5 40.5 RT (min) SDLAVPSELALLKYK spiked in urine samples XIC 10 ppm Transitions : 682.40->878.54 682.40->977.61 Gallien et al., J. Proteomics 2013 38.5 39.5 40.5 RT (min) Domon - 2015 Skline Webinar 17
Selectivit in HR/AM Mode /2 XIC 700 ppm XIC 10 ppm 38.5 39.5 40.5 RT (min) 38.5 39.5 40.5 RT (min) SDLAVPSELALLKYK spiked in urine samples Transitions 682.40 -> 878.54 xxx -> 878.45, Interference 682.40 -> 977.61 -> 977.52, Interference XIC 10 ppm 38.5 39.5 40.5 RT (min) Domon - 2015 Skline Webinar 18
Selectivit of PRM Measurements 1 2 Selection Window Width 3 [Tpicall: 1.0 (2.0) m/z High selectivit: 0.7 (0.4) m/z] (High performance Quad) High Resolution (OT) [35,000 140,000] Domon - 2015 Skline Webinar 19
Interfered transition (%) Selectivit of MS/MS Analses 60 50 40 30 20 10 0 2200 fragments analzed 25 16 8 4 Quadrupole isolation window (Th) 2 17500 35000 70000 Orbitrap resolution (@m/z 200) > Selectivit of measurements is affected b the precursor isolation window > Increased (nominal) orbitrap resolution (17 k to 70k) partiall compensate Gallien et al., J. Proteomics 2012 Domon - 2015 Skline Webinar 20
Conclusion o Parallel Reaction Monitoring o High-resolution accurate mass quantification is an alternative to conventional SRM o Simple experimental design o Acquisition and data analsis are decoupled o Onl precursor m/z and elution times are required a priori o Iterative data processing: selection of fragment ions post-acquisition o Data analsis is performed using conventional tools: Skline o Improved data qualit o High confidence assignments: accurate mass; reference MS/MS spectra) o Increased analtical precision: high selective measurements. Domon - 2015 Skline Webinar 21