Targeted Quantitative Proteomics Using Selected Reaction ...
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• Selected reaction monitoring (SRM)-MS coupled with stable isotope dilution is becoming a powerful tool for targeted quantitative proteomics, e.g., biomarker verification. • Selective and sensitive detection of specific peptides are achieved by selecting a specific parent ion and monitoring specific fragment ions (See Fig.1). 537.63 → 656.36 537.63 → 691.88 537.63 → 756.40 536.29 → 654.36 536.29 → 689.88 536.29 → 754.40 18 O labeled transitions 16 O residual transition Protein Sequence % of 16 O residual peptides* % CV (triplicate analyses) Carbonic anhydrase DGPLTGTYR 0.35 10.7 Carbonic anhydrase DFPIANGER 0.50 27.8 Carbonic anhydrase EPISVSSQQMLK 0.73 33.5 Carbonic anhydrase AVVQDPALKPLALVYGEATSR 0.15 10.1 β‐Lactoglobulin LIVTQTMK 0.29 35.8 β‐Lactoglobulin VLVLDTDYKK 0.06 4.2 β‐Lactoglobulin TPEVDDEALEK 2.27 13.2 β‐Lactoglobulin VYVEELKPTPEGDLEILLQK 0.20 13.0 Cytochrome c EDLIAYLK 0.09 37.7 Cytochrome c TGPNLHGLFGR 0.93 17.6 Myoglobin LFTGHPETLEK 0.57 14.0 Myoglobin VEADIAGHGQEVLIR 0.48 3.0 Ovalbumin AFKDEDTQAMPFR 0.05 21.6 Ovalbumin GGLEPINFQTAADQAR 0.25 34.5 β‐Galactosidase VDEDQPFPAVPK 0.35 32.8 β‐Galactosidase LWSAEIPNLYR 0.11 6.9 Targeted Quantitative Proteomics Using Selected Reaction Monitoring (SRM)- Mass Spectrometry Coupled with an 18 O-labeled Reference as Internal Standards Jong-Seo Kim, Errol Robinson, Boyd L. Champion, Brianne O. Petritis, Thomas L. Fillmore, Ronald J. Moore, Liu Tao, David G. Camp II, Richard D. Smith, and Wei-Jun Qian Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA Introduction Overview Methods Results Acknowledgements Portions of this work were supported by the NIH Director's New Innovator Award Program 1-DP2OD006668-01 (to W.J.Q.) and NIH National Center for Research Resources RR18522 (to R.D.S.) Experiment work was performed in the Environmental Molecular Science Laboratory, a DOE/BER national scientific user facility at Pacific Northwest National Laboratory (PNNL) in Richland, Washington. PNNL is operated for the DOE by Battelle under contract DE-AC05-76RLO-1830. References 1. Qian W-J, et al. Large-Scale Multiplexed Quantitative Discovery Proteomics Enabled by the Use of an 18 O-Labeled "Universal" Reference Sample. J Proteome Res 8:290-299 (2009). 2. Petritis BO et al. A Simple Procedure for Effective Quenching of Trypsin Activity and Prevention of 18 O-Labeling Back-Exchange. J Proteome Res 8:2157-2163 (2009). Conclusions 18 O labeling efficiency CONTACT: Jong-Seo Kim, Ph.D. Biological Sciences Division, K8-98 Pacific Northwest National Laboratory P.O. Box 999, Richland, WA 99352 E-mail: [email protected] • To address the need of stable isotope labeled internal standards for accurate quantification, we introduce the use of an 18 O-labeled reference as comprehensive internal standards for accurate SRM-MS- based quantification instead of using labeled synthetic peptides. • 18 O-labeling efficiency for most peptides is sufficiently high to provide a residue of 16 O/ 18 O ratio of <1%. • A linear dynamic range of quantification ~10 4 in relative concentration was obtained for most of the peptides being monitored based on the observed peak area ratios ( 16 O/ 18 O) versus concentration ratios for each peptide. • 16 O/ 18 O based quantification also provided significantly better reproducibility in terms of the coefficient of variances among the triplicate analyses compared to label-free quantification. • The utility of 18 O-labeled “universal” reference as internal standards for targeted quantitative proteomics has been successfully demonstrated – A linear dynamic range of quantification ~ 10 4 in relative concentration – Better reproducibility than label-free approach – Sufficient effectiveness for monitoring 16 O and 18 O y-fragments at unit or even higher resolution • Endogenous concentration of CRP and spiked PSA in human plasma successfully determined by “standard protein addition/ 18 O-labeled reference” Fig. 4 LC-TSQ-SRM chromatogram showing 16 O residual (top) and 18 O labeled (bottom) transitions of VDEDQPFPAVPK(3+) peptide after 18 O labeling. Fig. 1 SRM quantification strategy using 18 O-labeled “universal” reference. 1 LC-SRM-MS Patient samples mixed with 18 O-labeled reference Pooled reference sample Patient peptide samples LC-SRM-MS analysis Group A: Group B: Patient Samples Trypsin digestion Quantitative information based on 16 O/ 18 O peak area ratios 18 O labeling 18 O-labeled “universal” reference Fig. 2 Flow chart for preparing the non-depleted mouse plasma samples with 6 spiked standard protein digests for calibration curve. • Bovine carbonic anhydrase, beta-lactoglobulin, cytochrome c, chicken ovalbumin, equine skeletal muscle myoglobin and E.coli beta- galactosidase were used as 6 standard proteins. • C-reactive protein (CRP) and prostate specific antigen (PSA) were spiked in human female plasma (non-depleted) for absolute quantification using standard addition. • 18 O labeling was performed with the recently published protocol to completely prevent 18 O back-exchange. 2 • LC: Agilent 1100; 75 μm x 25 cm column (3 μm; C18); 60-min gradient; 400 nL/min. • MS: Thermo Scientific TSQ vantage. • SRM: 10 ms dwell time; 0.002 Da scan width; unit resolution in Q1 and Q3 (0.7 FWHM). • Pinpoint software (Thermo Fisher Scientific) was used for data analysis. Non-depleted mouse plasma digest (~40 mg/mL total protein) Add 6 standard protein digests into plasma at 0.001%, 0.01%, 0.1% and 1% (w/w) to total mouse plasma digest 18 O labeling 18 O-labeled reference 16 O-samples Combine 16 O-labeled samples and 18 O-labeled reference ( 16 O at 0, 5, 10, 25, 50, 100, 500 ng/mL per 18 O at 500 ng/mL, 16 O at 50, 250, 500, 1250, 2500, 5000 ng/mL per 18 O at 50 ng/mL) LC-SRM-MS analysis and standard calibration curve Select reference level Linear dynamic range of 18 O labeled reference–based quantification Effect of Q1 resolution Fig. 6 Calibration curve showing linear dynamic range of quantification ~10 4 in relative concentration. Red error bars: label-free peak area ratio (= each 16 O area / average 18 O area) Green error bars: each pair area ratio of 16 O/ 18 O Concen. Ratio 0.01 0.02 0.04 0.1 0.2 1 5 10 25 50 100 16 O/ 18 O pairs 6.1 12.5 13.7 13.8 5.5 7.7 16.8 2.8 4.6 4.2 1.6 Label free (%) 39.2 23.4 47.5 26.5 19.3 10.6 31.2 3.2 10.7 4.8 3.5 Absolute quantification by standard addition Fig. 3 LC-LTQ-Orbitrap MS analysis of 18 O labeled peptides. Fig. 8 Standard addition calibration curve for absolute quantification of endogenous CRP and spiked PSA in non- depleted human female plasma. Reproducibility Fig. 9 XIC of 16 O/ 18 O transition pair for endogenous CRP (left) and spiked PSA (right) at 500 ng/mL in non-depleted human female plasma. 354.71 →490.30 356.71 →494.31 379.25 →458.31 381.25 →462.32 Area ratio 0.37 Area ratio 0.33 • To achieve accurate quantification, isotope labeled synthetic peptides are often used as internal standards, which limits the feasibility to monitor a large number of candidates due to expense. • The 18 O-labeled “universal” reference will serve as ideal internal standards for quantitative monitoring for any candidates of interest. Product ion chromatogram Q1 Triple Quadrupole MS (Selected Reaction Monitoring Mode) Detector LC-ESI Q2 Q3 Precursor ion selection Product ion selection Product ion chromatogram Time (min) Product ion chromatogram Q1 Triple Quadrupole MS (Selected Reaction Monitoring Mode) Detector LC-ESI Q2 Q3 Precursor ion selection Product ion selection Q1 Triple Quadrupole MS (Selected Reaction Monitoring Mode) Detector LC-ESI Q2 Q3 Precursor ion selection Product ion selection (354.71→490.30)/ (356.71→494.31) Standard added CRP (μg/mL) C-Reactive Protein (AFVFPK) 16 O/ 18 O area ratio (379.25→458.31)/ (381.25→464.32) Standard added PSA (μg/mL) Prostate Specific Antigen (SVILLGR) 16 O/ 18 O area ratio Table 1. Percentage of 16 O residual (unlabeled) peptide in an 18 O-labeled reference samples determined by SRM mode * An average of ~0.5% 16 O/ 18 O ratio indicates the high percentage of heavy label incorporation, a necessity for accurate quantification. Fig. 5 Effect of Q1 window width on y type ions. 16 O / 18 O y ion pair without interference under wide Q1 window. (Da) Concentration ratio ( 16 O/ 18 O) 16 O/ 18 O area ratio 16 O/ 18 O area ratio 16 O/ 18 O area ratio Fig. 7 Comparison of reproducibility (% CV in table) between 16 O/ 18 O pair and label free ( 16 O/ 18 O avg ) approach. Table 2. SRM Quantification results of endogenous CRP and spiked PSA (500 ng/mL) in non-depleted human female plasma by standard addition method. CRP (ng/mL) (endogenous) (354.71→ 490.30) / (356.71→494.31) concentration CV 790 ng/mL 4.1% PSA (ng/mL) (spiked) (379.25→458.31) / (381.25→464.32) concentration CV 800 ng/mL 0.8% Concentration ratio ( 16 O/ 18 O) 16 O/ 18 O area ratio 16 O/ 18 O area ratio
Transcript of Targeted Quantitative Proteomics Using Selected Reaction ...
• Selected reaction monitoring (SRM)-MS coupled with stable isotope dilution is becoming a powerful tool for targeted quantitative proteomics, e.g., biomarker verification.
• Selective and sensitive detection of specific peptides are achieved by selecting a specific parent ion and monitoring specific fragment ions (See Fig.1).
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Protein Sequence% of 16O residual peptides*
% CV(triplicate analyses)
Carbonic anhydrase DGPLTGTYR 0.35 10.7
Carbonic anhydrase DFPIANGER 0.50 27.8
Carbonic anhydrase EPISVSSQQMLK 0.73 33.5
Carbonic anhydrase AVVQDPALKPLALVYGEATSR 0.15 10.1
β‐Lactoglobulin LIVTQTMK 0.29 35.8
β‐Lactoglobulin VLVLDTDYKK 0.06 4.2
β‐Lactoglobulin TPEVDDEALEK 2.27 13.2
β‐Lactoglobulin VYVEELKPTPEGDLEILLQK 0.20 13.0
Cytochrome c EDLIAYLK 0.09 37.7
Cytochrome c TGPNLHGLFGR 0.93 17.6
Myoglobin LFTGHPETLEK 0.57 14.0
Myoglobin VEADIAGHGQEVLIR 0.48 3.0
Ovalbumin AFKDEDTQAMPFR 0.05 21.6
Ovalbumin GGLEPINFQTAADQAR 0.25 34.5
β‐Galactosidase VDEDQPFPAVPK 0.35 32.8
β‐Galactosidase LWSAEIPNLYR 0.11 6.9
Targeted Quantitative Proteomics Using Selected Reaction Monitoring (SRM)-Mass Spectrometry Coupled with an 18O-labeled Reference as Internal Standards Jong-Seo Kim, Errol Robinson, Boyd L. Champion, Brianne O. Petritis, Thomas L. Fillmore, Ronald J. Moore, Liu Tao, David G. Camp II, Richard D. Smith, and Wei-Jun QianBiological Sciences Division, Pacific Northwest National Laboratory, Richland, WA
Introduction
Overview
Methods Results
AcknowledgementsPortions of this work were supported by the NIH Director's New Innovator Award Program 1-DP2OD006668-01 (to W.J.Q.) and NIH National Center for Research Resources RR18522 (to R.D.S.) Experiment work was performed in the Environmental Molecular Science Laboratory, a DOE/BER national scientific user facility at Pacific Northwest National Laboratory (PNNL) in Richland, Washington. PNNL is operated for the DOE by Battelle under contract DE-AC05-76RLO-1830.
References1. Qian W-J, et al. Large-Scale Multiplexed Quantitative Discovery
Proteomics Enabled by the Use of an 18O-Labeled "Universal" Reference Sample. J Proteome Res 8:290-299 (2009).
2. Petritis BO et al. A Simple Procedure for Effective Quenching ofTrypsin Activity and Prevention of 18O-Labeling Back-Exchange. J Proteome Res 8:2157-2163 (2009).
Conclusions18O labeling efficiency
CONTACT: Jong-Seo Kim, Ph.D.Biological Sciences Division, K8-98Pacific Northwest National LaboratoryP.O. Box 999, Richland, WA 99352E-mail: [email protected]
• To address the need of stable isotope labeled internal standards for accurate quantification, we introduce the use of an 18O-labeled reference as comprehensive internal standards for accurate SRM-MS-based quantification instead of using labeled synthetic peptides.
• 18O-labeling efficiency for most peptides is sufficiently high to provide a residue of 16O/18O ratio of <1%.
• A linear dynamic range of quantification ~104
in relative concentration was obtained for most of the peptides being monitored based on the observed peak area ratios (16O/18O) versus concentration ratios for each peptide.
• 16O/18O based quantification also provided significantly better reproducibility in terms of the coefficient of variances among the triplicate analyses compared to label-free quantification.
• The utility of 18O-labeled “universal”reference as internal standards for targeted quantitative proteomics has been successfully demonstrated– A linear dynamic range of quantification
~ 104 in relative concentration– Better reproducibility than label-free
approach– Sufficient effectiveness for monitoring
16O and 18O y-fragments at unit or even higher resolution
• Endogenous concentration of CRP and spiked PSA in human plasma successfully determined by “standard protein addition/18O-labeled reference”
Fig. 4 LC-TSQ-SRM chromatogram showing 16O residual (top) and 18O labeled (bottom) transitions of VDEDQPFPAVPK(3+) peptide after 18O labeling.
Fig. 1 SRM quantification strategy using 18O-labeled “universal” reference.1
LC-SRM-MS
Patient samples mixed with 18O-labeled reference
Pooled reference sample
Patient peptide samples
LC-SRM-MS analysis
Group A:Group B:
Patient Samples
Trypsin digestion
Quantitative information based on 16O/18O peak area ratios
18O labeling
18O-labeled “universal”reference
Fig. 2 Flow chart for preparing the non-depleted mouse plasma samples with 6 spiked standard protein digests for calibration curve.
• Bovine carbonic anhydrase, beta-lactoglobulin, cytochrome c, chicken ovalbumin, equine skeletal muscle myoglobin and E.coli beta-galactosidase were used as 6 standard proteins.
• C-reactive protein (CRP) and prostate specific antigen (PSA) were spiked in human female plasma (non-depleted) for absolute quantification using standard addition.
• 18O labeling was performed with the recently published protocol tocompletely prevent 18O back-exchange.2
• LC: Agilent 1100; 75 μm x 25 cm column (3 μm; C18); 60-min gradient; 400 nL/min.
• MS: Thermo Scientific TSQ vantage.• SRM: 10 ms dwell time; 0.002 Da scan width; unit resolution in Q1 and
Q3 (0.7 FWHM).• Pinpoint software (Thermo Fisher Scientific) was used for data analysis.
Non-depleted mouse plasma digest(~40 mg/mL total protein)
Add 6 standard protein digests into plasma at 0.001%, 0.01%, 0.1% and 1% (w/w) to total mouse plasma digest
18O labeling
18O-labeled reference16O-samples
Combine 16O-labeled samples and 18O-labeled reference(16O at 0, 5, 10, 25, 50, 100, 500 ng/mL per 18O at 500 ng/mL,
16O at 50, 250, 500, 1250, 2500, 5000 ng/mL per 18O at 50 ng/mL)
LC-SRM-MS analysis and standard calibration curve
Select reference level
Linear dynamic range of 18O labeled reference–based quantification
Effect of Q1 resolution
Fig. 6 Calibration curve showing linear dynamic range of quantification ~104 in relative concentration.
Red error bars: label-free peak area ratio (= each 16O area / average 18O area)Green error bars: each pair area ratio of 16O/18O
Concen. Ratio
0.01 0.02 0.04 0.1 0.2 1 5 10 25 50 100
16O/18Opairs
6.1 12.5 13.7 13.8 5.5 7.7 16.8 2.8 4.6 4.2 1.6
Label free (%)
39.2 23.4 47.5 26.5 19.3 10.6 31.2 3.2 10.7 4.8 3.5
Absolute quantification by standard addition
Fig. 3 LC-LTQ-Orbitrap MS analysis of 18O labeled peptides.
Fig. 8 Standard addition calibration curve for absolute quantification of endogenous CRP and spiked PSA in non-depleted human female plasma.
Reproducibility
Fig. 9 XIC of 16O/18O transition pair for endogenous CRP (left) and spiked PSA (right) at 500 ng/mL in non-depleted human female plasma.
354.71→490.30
356.71→494.31
379.25→458.31
381.25→462.32
Area ratio0.37
Area ratio0.33
• To achieve accurate quantification, isotope labeled synthetic peptides are often used as internal standards, which limits the feasibility to monitor a large number of candidates due to expense.
• The 18O-labeled “universal” reference will serve as ideal internal standards for quantitative monitoring for any candidates of interest.
Time (min)
Product ion chromatogram
Q1
Triple Quadrupole MS(Selected Reaction Monitoring Mode)
DetectorLC-ESI
Q2 Q3
Precursor ion
selection
Product ion
selection Time (min)
Product ion chromatogram
Time (min)
Product ion chromatogram
Q1
Triple Quadrupole MS(Selected Reaction Monitoring Mode)
DetectorLC-ESI
Q2 Q3
Precursor ion
selection
Product ion
selection
Q1
Triple Quadrupole MS(Selected Reaction Monitoring Mode)
DetectorLC-ESI
Q2 Q3
Precursor ion
selection
Product ion
selection
(354.71→490.30)/(356.71→494.31)
Standard added CRP (μg/mL)
C-Reactive Protein(AFVFPK)
16O
/18 O
are
a ra
tio
(379.25→458.31)/(381.25→464.32)
Standard added PSA (μg/mL)
Prostate Specific Antigen(SVILLGR)
16O
/18 O
are
a ra
tio
Table 1. Percentage of 16O residual (unlabeled) peptide in an 18O-labeled reference samples determined by SRM mode
* An average of ~0.5% 16O/18O ratio indicates the high percentage of heavy label incorporation, a necessity for accurate quantification.
Fig. 5 Effect of Q1 window width on y type ions. 16O / 18O y ion pair without interference under wide Q1 window.
(Da)
Concentration ratio (16O/18O)
16O
/18O
are
a ra
tio16
O/18
O a
rea
ratio
16O
/18O
are
a ra
tio
Fig. 7 Comparison of reproducibility (% CV in table) between 16O/18O pair and label free (16O/18Oavg) approach.
Table 2. SRM Quantification results of endogenous CRP and spiked PSA (500 ng/mL) in non-depleted human female plasma by standard addition method.
CRP (ng/mL)(endogenous)
(354.71→ 490.30) / (356.71→494.31)concentration CV790 ng/mL 4.1%
PSA (ng/mL)(spiked)
(379.25→458.31) / (381.25→464.32)concentration CV800 ng/mL 0.8%
Concentration ratio (16O/18O)
16O
/18O
are
a ra
tio
16O
/18O
are
a ra
tio
