Target Analyses in Parallel Reaction Monitoring Mode (PRM) Skyline Webinar January 13, 2015 Bruno Domon, PhD Head Luxembourg Clinical Proteomics Center Invited Professor University of Luxembourg

Introduction Target Quantitative Assays Domon - 2015 Skyline Webinar

Characteristics of Quantitative Assays Sensitivity Selectivity Scale Detection of abundance components Wide range of protein concentrations Need for low LoD / LoQ Wide dynamic range Complexity of proteomic samples Reduce sample complexity (interferences in measurements) High resolution instruments: LC + MS Biological variability Need to perform large studies Throughput, i.e. robust platform Multiplexing capability Domon - 2015 Skyline Webinar

Types of Targeted Experiments Sensitivity Selectivity Scale Classical Quantitative Experiment Precise quantification (biomarkers) Internal standards (calibrated amount) Limited number of analytes Screening Experiment Detection of peptides in complex matrix (e.g. blood or urine samples) Large scale (hundred of candidates) Multiplexing capability Sensitivity Selectivity Scale Gallien et al., J. Mass Spectrom. 2011 Domon - 2015 Skyline Webinar 4

Selected Reaction Monitoring (SRM) Triple quadrupole instrument min Quantification (Traces; AUC) m/z 513 827 975 1018 789 Identity confirmation Kim et al., Proteomics Clin. Appl. 2013 Domon - 2015 Skyline Webinar

Targeted Proteomics SRM experiments: triple quadrupole instrument - reference method Q3 Q1 CID Fixed Limitations: Actual number of transitions to be monitored Low resolution mass analyzers (both Q1 and Q3) > co-isolation of interferences along with the precursor ion Signal observed Interference Analyte [m/z] Gallien et al., J. Mass Spectrom. 2011 Domon - 2015 Skyline Webinar

Selectivity is an Issue ….. Expected Observed "One train can hide another one ! " Check twice ! Domon - 2015 Skyline Webinar

Selectivity of Measurements SRM 5E+04 Intensity (coutns/s) 22 24 26 28 Elution time (min) NLLSVAYK NLLSVAYK y3+ y4+ y5+ y6+ 4E+05 3E+05 2E+05 1E+05 Intensity (coutns/s) 25 27 29 31 Elution time (min) PRM NLLSVAYK NLLSVAYK 8E+03 8E+03 y3+ y4+ y5+ y6+ y3+ y4+ y5+ y6+ 6E+03 6E+03 Intensity (coutns/s) Intensity (coutns/s) 4E+03 4E+03 2E+03 2E+03 22 24 26 28 25 27 29 31 Elution time (min) Elution time (min) Domon - 2015 Skyline Webinar Kim et al., Proteomics Clin. Appl. 2013

Parallel Reaction Monitoring (PRM) Domon - 2015 Skyline Webinar

Parallel Reaction Monitoring (PRM) Performed on a quadrupole / orbitrap instrument (high-resolution) Source MS-1 CID MS-2 Fixed m/z Simplified experimental design Selection of transitions, extraction of traces post-acquisition. time Data Analysis Gallien et al., J. Proteomics 2014 Domon - 2015 Skyline Webinar

Design of a Targeted Experiment: PRM Mode Quadrupole – Orbitrap / PRM mode Simplified Method Design Flexible Data Analysis Peptide Selection Transition Selection Optimization Peptides > Protein_1 > Protein_2 > Protein_i Domon - 2015 Skyline Webinar

Parallel Reaction Monitoring Mode (PRM) Isolation Fragmentation 3. Transfer in OT 4. HR Analysis Domon - 2015 Skyline Webinar

Parallel Reaction Monitoring Experiment 1 2 3 PRM mode (multiplexing capability) Full MS/MS Spectra Q-orbitrap instrument m/z 535 827 975 1018 513 789 min 513 975 789 Ion Chrom. extraction Fingerprint to confirm the analyte identity Chrom. traces for quantification Kim et al., Proteomics Clin. Appl. 2013 Domon - 2015 Skyline Webinar

Quantification Methods in PRM Sequential: Iterative analyses Sequential isolation / fragmentation events Multiple detection scans t Pep1 Pep2 Pep3 Multiplexed: Parallel analysis Sequential isolation and fragmentation Intermediate storage Single detection scan Pep1, Pep2, Pep3 t Isolation (Q) Detection of fragments (OT) Fragmentation (Coll Cell) Gallien et al., Mol. Cell. Proteomics 2012 Domon - 2014 Skyline Webinar

PRM Mode: Multiplexed Analysis L H Sequential isolation of L/H precursors Fragmentation and storage in HCD cell One orbitrap detection scan Quantification based fragment ions Selection of ions post-acquisition y2+ y4+ y5+ y6+ y7+ y8+ y3+ EGYYGYTGAFR (L) / EGYYGYTGAFR (H) 31 32 L 31 32 H Quantification similar to SRM, but using high resolution fragment ions Domon - 2015 Skyline Webinar

PRM Analyses of Plasma Samples  Poor correlation SRM Data Peptides FIIPQIVK and LIAPVAEEEATVPNNK Surrogates: L-lactate dehydrogenase Plasma samples (Alb+IgG depleted) Pilot study: 3 controls and 3 disease  Excellent consistency PRM Data Domon - 2015 Skyline Webinar

Selectivity in HR/AM Mode /1 SRM on Triple quadrupole PRM on Q-Orbitrap XIC 700 ppm Res Q3 = 0.7 XIC 10 ppm SDLAVPSELALLKYK spiked in urine samples Transitions : 682.40->878.54 682.40->977.61 Gallien et al., J. Proteomics 2013 Domon - 2015 Skyline Webinar

Selectivity in HR/AM Mode /2 XIC 700 ppm XIC 10 ppm SDLAVPSELALLKYK spiked in urine samples XIC 10 ppm Transitions 682.40 -> 878.54 xxx -> 878.45 , Interference 682.40 -> 977.61 yyy -> 977.52, Interference Domon - 2015 Skyline Webinar

Selectivity of PRM Measurements 1 2 3 Selection Window Width [Typically: 1.0 (2.0) m/z High selectivity: 0.7 (0.4) m/z] (High performance Quad) High Resolution (OT) [35,000 – 140,000] Domon - 2015 Skyline Webinar

Selectivity of MS/MS Analyses 2200 fragments analyzed > Selectivity of measurements is affected by the precursor isolation window > Increased (nominal) orbitrap resolution (17 k to 70k) partially compensate Gallien et al., J. Proteomics 2012 Domon - 2015 Skyline Webinar

Conclusion Parallel Reaction Monitoring High-resolution accurate mass quantification is an alternative to conventional SRM Simple experimental design Acquisition and data analysis are decoupled Only precursor m/z and elution times are required a priori Iterative data processing: selection of fragment ions post-acquisition Data analysis is performed using conventional tools: Skyline Improved data quality High confidence assignments: accurate mass; reference MS/MS spectra) Increased analytical precision: high selective measurements. Domon - 2015 Skyline Webinar