期刊
JOURNAL OF PROTEOME RESEARCH
卷 10, 期 9, 页码 4334-4341出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr200156b
关键词
ion trap; CID; HCD; triple quadrupole; selected reaction monitoring; spectral correlation
资金
- European Union [262067]
- CTMM, the Center for Translational Molecular Medicine [01C-102]
- Netherlands Heart Foundation
- Netherlands Proteomics Centre
- Centre for Biomedical Genetics
- Netherlands Genomics Initiative
In proteomics, selected reaction monitoring (SRM) is rapidly gaining importance for targeted protein quantification. The triple quadrupole mass analyzers used in SRM assays allow for levels of specificity and sensitivity hard to accomplish by more standard shotgun proteomics experiments. Often, an SRM assay is built by in silica prediction of transitions and/or extraction of peptide precursor and fragment ions from a spectral library. Spectral libraries are typically generated from nonideal ion trap based shotgun proteomics experiments or synthetic peptide libraries, consuming considerable time and effort. Here, we investigate the usability of beam type CID (or higher energy CID (HCD)) peptide fragmentation spectra, as acquired using an Orbitrap Velos, to facilitate SRM assay development. Therefore, peptide fragmentation spectra, obtained by ion-trap CID, triple-quadrupole CM (QqQ-CID) and Orbitrap HCD, originating from digested cellular lysates, were compared. Spectral comparison and a dedicated correlation algorithm indicated significantly higher similarity between QqQ-CID and HCD fragmentation spectra than between QqQ-CID and ion trap-CID spectra. SRM transitions generated using a constructed HCD spectral library increased SRM assay sensitivity up to 2-fold, when compared to the use of a library created from more conventionally used ion trap-CID spectra, showing that HCD spectra can assist SRM assay development.
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