1. 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

2. Introduction Target Quantitative Assays
Domon
Skyline Webinar

3. 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
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4. 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
Skyline Webinar 4

5. 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
Skyline Webinar

6. 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
Skyline Webinar

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

8. 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
Skyline Webinar
Kim et al., Proteomics Clin. Appl. 2013

9. Parallel Reaction Monitoring (PRM)
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10. 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
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Skyline Webinar

11. 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
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12. Parallel Reaction Monitoring Mode (PRM)
Isolation
Fragmentation
3. Transfer in OT
4. HR Analysis
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13. Parallel Reaction Monitoring Experiment1 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
Skyline Webinar

14. 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
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15. 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) L H
Quantification similar to SRM, but using high resolution fragment ions
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16. 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
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17. 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 : >878.54
>977.61
Gallien et al., J. Proteomics 2013
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18. Selectivity in HR/AM Mode /2
XIC 700 ppm
XIC 10 ppm
SDLAVPSELALLKYK
spiked in urine samples
XIC 10 ppm
Transitions
>
xxx > , Interference
>
yyy > , Interference
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Skyline Webinar

19. Selectivity of PRM Measurements1 2 3
Selection Window
Width
[Typically: (2.0) m/z
High selectivity: (0.4) m/z]
(High performance Quad)
High Resolution (OT)
[35,000 – 140,000]
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20. 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
Skyline Webinar

21. 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
Skyline Webinar