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Title: 18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry

Selected reaction monitoring-mass spectrometry (SRM-MS) is an emerging technology for high throughput targeted protein quantification and verification in biological and biomarker discovery studies; however, the cost associated with the use of stable isotope labeled synthetic peptides as internal standards is prohibitive for quantitatively screening large numbers of candidate proteins as often required in the pre-verification phase of biomarker discovery. Herein we present the proof-of-concept experiments of using an 18O-labeled 'universal' reference as comprehensive internal standards for quantitative SRM-MS analysis. With an 18O-labeled whole proteome sample as reference, every peptide of interest will have its own corresponding heavy isotope labeled internal standard, thus providing an ideal approach for quantitative screening of a large number of candidates using SRM-MS. Our results showed that the 18O incorporation efficiency using a recently improved protocol was >99.5% for most peptides investigated, a level comparable to 13C/15N labeled synthetic peptides in terms of heavy isotope incorporation. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into mouse plasma with an 18O-labeled mouse plasma reference. A dynamic range of four orders of magnitude in relative concentration was obtained with high reproducibility (i.e., coefficient of variance <10%)more » based on the 16O/18O peak area ratios. Absolute and relative quantification of C-reactive protein and prostate-specific antigen were demonstrated by coupling an 18O-labeled reference with standard additions of protein standards. Collectively, our results demonstrated that the use of 18O-labeled reference provides a convenient and effective strategy for quantitative SRM screening of large number of candidate proteins.« less
Authors:
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Publication Date:
OSTI Identifier:
1033060
Report Number(s):
PNNL-SA-77028
40991; 400412000; TRN: US201202%%562
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Molecular and Cellular Proteomics; Journal Volume: 10; Journal Issue: 12
Research Org:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; ACCURACY; ANTIGENS; EFFICIENCY; GLOBULINS; IMMUNITY; INTERNET; PEPTIDES; PLASMA; PROTEINS; SPECTROSCOPY; STABLE ISOTOPES; VERIFICATION; selected reaction monitoring; multiple reaction monitoring; targeted proteomics; 18 O-labeling; "universal" reference; internal standard; protein standard; absolute quantification; Environmental Molecular Sciences Laboratory