skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Large-Scale Multiplexed Quantitative Discovery Proteomics Enabled by the Use of an O-18-Labeled “Universal” Reference Sample

Abstract

Quantitative comparison of protein abundances across a relatively large number of patient samples is an important challenge for clinical proteomic applications. Herein we describe a dual-quantitation strategy that allows the simultaneous integration of complementary label-free and stable isotope labeling based approaches without increasing the number of LC-MS analyses. The approach utilizes a stable isotope 18O-labeled “universal” reference sample as a comprehensive set of internal standards spiked into each individually processed unlabeled patient sample. The quantitative data are based on both the direct 16O-MS intensities for label-free quantitation and the 16O/18O isotopic peptide pair ratios that compare each patient sample to the identical labeled reference. The effectiveness of this dual-quantitation approach for large scale quantitative proteomics is demonstrated by the application to a set of 38 clinical plasma samples from surviving and non-surviving severe burn patients. With the coupling of immunoaffinity depletion, cysteinyl-peptide enrichment based fractionation, high resolution LC-MS measurements, and the dual-quantitation approach, a total of 318 proteins were confidently quantified with at least two peptides and 263 proteins were quantified by both approaches. The strategy also enabled a direct comparison between the two approaches with the labeling approach showing significantly better precision in quantitation while the label-free approach resultedmore » in more protein identifications. The relative abundance differences determined by the two approaches also show strong correlation. Finally, the dual-quantitation strategy allowed us to identify more candidate protein biomarkers, illustrating the complementary nature of the two quantitative methods.« less

Authors:
; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
949081
Report Number(s):
PNNL-SA-57317
24698; TRN: US200907%%390
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Journal of Proteome Research, 8(1):290-299
Additional Journal Information:
Journal Volume: 8; Journal Issue: 1
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ABUNDANCE; ACCURACY; FRACTIONATION; PATIENTS; PEPTIDES; PLASMA; PROTEINS; RESOLUTION; STABLE ISOTOPES; Environmental Molecular Sciences Laboratory

Citation Formats

Qian, Weijun, Liu, Tao, Petyuk, Vladislav A, Gritsenko, Marina A, Petritis, Brianne O, Polpitiya, Ashoka D, Kaushal, Amit, Xiao, Wenzhong, Finnerty, Celeste C, Jescheke, Marc G, Jaitly, Navdeep, Monroe, Matthew E, Moore, Ronald J, Moldawer, Lyle L, Davis, Ronald W, Tompkins, Ronald G, Hemdon, David N, Camp, David G, and Smith, Richard D. Large-Scale Multiplexed Quantitative Discovery Proteomics Enabled by the Use of an O-18-Labeled “Universal” Reference Sample. United States: N. p., 2009. Web. doi:10.1021/pr800467r.
Qian, Weijun, Liu, Tao, Petyuk, Vladislav A, Gritsenko, Marina A, Petritis, Brianne O, Polpitiya, Ashoka D, Kaushal, Amit, Xiao, Wenzhong, Finnerty, Celeste C, Jescheke, Marc G, Jaitly, Navdeep, Monroe, Matthew E, Moore, Ronald J, Moldawer, Lyle L, Davis, Ronald W, Tompkins, Ronald G, Hemdon, David N, Camp, David G, & Smith, Richard D. Large-Scale Multiplexed Quantitative Discovery Proteomics Enabled by the Use of an O-18-Labeled “Universal” Reference Sample. United States. https://doi.org/10.1021/pr800467r
Qian, Weijun, Liu, Tao, Petyuk, Vladislav A, Gritsenko, Marina A, Petritis, Brianne O, Polpitiya, Ashoka D, Kaushal, Amit, Xiao, Wenzhong, Finnerty, Celeste C, Jescheke, Marc G, Jaitly, Navdeep, Monroe, Matthew E, Moore, Ronald J, Moldawer, Lyle L, Davis, Ronald W, Tompkins, Ronald G, Hemdon, David N, Camp, David G, and Smith, Richard D. 2009. "Large-Scale Multiplexed Quantitative Discovery Proteomics Enabled by the Use of an O-18-Labeled “Universal” Reference Sample". United States. https://doi.org/10.1021/pr800467r.
@article{osti_949081,
title = {Large-Scale Multiplexed Quantitative Discovery Proteomics Enabled by the Use of an O-18-Labeled “Universal” Reference Sample},
author = {Qian, Weijun and Liu, Tao and Petyuk, Vladislav A and Gritsenko, Marina A and Petritis, Brianne O and Polpitiya, Ashoka D and Kaushal, Amit and Xiao, Wenzhong and Finnerty, Celeste C and Jescheke, Marc G and Jaitly, Navdeep and Monroe, Matthew E and Moore, Ronald J and Moldawer, Lyle L and Davis, Ronald W and Tompkins, Ronald G and Hemdon, David N and Camp, David G and Smith, Richard D},
abstractNote = {Quantitative comparison of protein abundances across a relatively large number of patient samples is an important challenge for clinical proteomic applications. Herein we describe a dual-quantitation strategy that allows the simultaneous integration of complementary label-free and stable isotope labeling based approaches without increasing the number of LC-MS analyses. The approach utilizes a stable isotope 18O-labeled “universal” reference sample as a comprehensive set of internal standards spiked into each individually processed unlabeled patient sample. The quantitative data are based on both the direct 16O-MS intensities for label-free quantitation and the 16O/18O isotopic peptide pair ratios that compare each patient sample to the identical labeled reference. The effectiveness of this dual-quantitation approach for large scale quantitative proteomics is demonstrated by the application to a set of 38 clinical plasma samples from surviving and non-surviving severe burn patients. With the coupling of immunoaffinity depletion, cysteinyl-peptide enrichment based fractionation, high resolution LC-MS measurements, and the dual-quantitation approach, a total of 318 proteins were confidently quantified with at least two peptides and 263 proteins were quantified by both approaches. The strategy also enabled a direct comparison between the two approaches with the labeling approach showing significantly better precision in quantitation while the label-free approach resulted in more protein identifications. The relative abundance differences determined by the two approaches also show strong correlation. Finally, the dual-quantitation strategy allowed us to identify more candidate protein biomarkers, illustrating the complementary nature of the two quantitative methods.},
doi = {10.1021/pr800467r},
url = {https://www.osti.gov/biblio/949081}, journal = {Journal of Proteome Research, 8(1):290-299},
number = 1,
volume = 8,
place = {United States},
year = {Thu Jan 01 00:00:00 EST 2009},
month = {Thu Jan 01 00:00:00 EST 2009}
}