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Title: Expediting SRM assay development for large-scale targeted proteomics experiments

Due to their high sensitivity and specificity, targeted proteomics measurements, e.g. selected reaction monitoring (SRM), are becoming increasingly popular for biological and translational applications. Selection of optimal transitions and optimization of collision energy (CE) are important assay development steps for achieving sensitive detection and accurate quantification; however, these steps can be labor-intensive, especially for large-scale applications. Herein, we explored several options for accelerating SRM assay development evaluated in the context of a relatively large set of 215 synthetic peptide targets. We first showed that HCD fragmentation is very similar to CID in triple quadrupole (QQQ) instrumentation, and by selection of top six y fragment ions from HCD spectra, >86% of top transitions optimized from direct infusion on QQQ instrument are covered. We also demonstrated that the CE calculated by existing prediction tools was less accurate for +3 precursors, and a significant increase in intensity for transitions could be obtained using a new CE prediction equation constructed from the present experimental data. Overall, our study illustrates the feasibility of expediting the development of larger numbers of high-sensitivity SRM assays through automation of transitions selection and accurate prediction of optimal CE to improve both SRM throughput and measurement quality.
Authors:
 [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [2] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1] ;  [1]
  1. Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Biological Sciences Division
  2. Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
Publication Date:
OSTI Identifier:
1167647
Report Number(s):
PNNL-SA--102906
Journal ID: ISSN 1535-3893; 40991; 46206; 48135; 400412000
Grant/Contract Number:
AC05-76RL01830; U24-CA-160019; P41GM103493; DP2OD006668
Type:
Accepted Manuscript
Journal Name:
Journal of Proteome Research
Additional Journal Information:
Journal Volume: 13; Journal Issue: 10; Journal ID: ISSN 1535-3893
Publisher:
American Chemical Society (ACS)
Research Org:
Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org:
National Inst. of Health (NIH); USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES CE prediction; HCD; MRM; optimization; QQQ; SRM; targeted quantification; transition selection; Environmental Molecular Sciences Laboratory