Target Analyses in Parallel Reaction Monitoring Mode (PRM)

Size: px

Start display at page:

Download "Target Analyses in Parallel Reaction Monitoring Mode (PRM)"

Cassandra Logan
6 years ago
Views:

1 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

2 INTRODUCTION TARGET QUANTITATIVE ASSAYS Domon Skline Webinar 2

3 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 Skline Webinar 3

4 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 Scale Selectivit Domon Skline Webinar 4

5 Selected Reaction Monitoring (SRM) Triple quadrupole instrument Quantification (Traces; AUC) min Identit confirmation m/z Kim et al., Proteomics Clin. Appl Domon Skline Webinar 5

6 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 Domon Skline Webinar 6

7 Selectivit is an Issue.. Expected Retention time (min) Observed "One train can hide another one! " Check twice! Retention time (min) Domon Skline Webinar 7

8 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.E Intensit (counts/s) 3E05 3.E05 2E05 2.E05 1E05 1.E E Elution Retention time time (min) (min) 0.E Retention Elution time time (min) (min) 8E03 8.E03 NLLSVAYK 8E03 8.E03 NLLSVAYK 6E03 6.E03 4E03 4.E E03 6.E03 4E03 4.E E03 2.E03 2E03 2.E03 0.E00 0.E Elution Retention time time (min) (min) Retention Elution time time (min) (min) Kim et al., Proteomics Clin. Appl Domon Skline Webinar 8

9 PARALLEL REACTION MONITORING (PRM) Domon Skline Webinar 9

10 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 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

11 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 Pep_A Pep_A Pep_A Pep_B Pep_B Pep_B Pep_B Pep_N Pep_N Pep_N Pep_N Domon Skline Webinar 11

12 Parallel Reaction Monitoring Mode (PRM) 1. Isolation 2. Fragmentation 3. Transfer in OT 4. HR Analsis Domon Skline Webinar 12

13 Parallel Reaction Monitoring Experiment 1 2 PRM mode (multiplexing capabilit) Q-orbitrap instrument 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 Kim et al., Proteomics Clin. Appl Domon Skline Webinar 13

14 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 Skline Webinar 14

15 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: AV: 1 NL: 8.40E5 Q11_0908 #8001 RT: AV: 1 F: NL: FTMS 8.40E5 p NSI Full msx ms @hcd @hcd29.13 [ ] F: FTMS p NSI Full msx ms @hcd @hcd29.13 [ ] EGYYGYTGAFR (L) / 80 EGYYGYTGAFR (H) o Quantification based fragment ions o Selection of ions post-acquisition L H m/z m/z Relative Abundance 9.00 Quantification similar to SRM, but using high resolution fragment ions L Area ratio H Retention Time (min) 32 Retention Time (min) Retention Time (min) Amount (fm Domon Skline Webinar 15

16 Fold change Fold change Fold change PRM Analses of Plasma Samples 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 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 Skline Webinar 16

17 Selectivit in HR/AM Mode /1 SRM on Triple quadrupole PRM on Q-Orbitrap Res Q3 = 0.7 XIC 700 ppm RT (min) RT (min) SDLAVPSELALLKYK spiked in urine samples XIC 10 ppm Transitions : > > Gallien et al., J. Proteomics RT (min) Domon Skline Webinar 17

18 Selectivit in HR/AM Mode /2 XIC 700 ppm XIC 10 ppm RT (min) RT (min) SDLAVPSELALLKYK spiked in urine samples Transitions > xxx -> , Interference > > , Interference XIC 10 ppm RT (min) Domon Skline Webinar 18

19 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] Domon 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) >

20 Interfered transition (%) Selectivit of MS/MS Analses fragments analzed Quadrupole isolation window (Th) 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 Skline Webinar 20

21 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 Skline Webinar 21

Amadeo R. Fernández-Alba

% of compounds % of compounds % of compounds % of compounds Amadeo R. Fernández-Alba LC-Orbitrap QExactive Focus Instrumental LOQ 1% 9% 8% 7% 6% 5% 4% 3% 2% 1% %.1 mg/g.2 mg/g Tomato.5 mg/g ddms2 Target