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Simplification of complex peptide mixtures for proteomic analysis: Reversible biotinylation of cysteinyl peptides
Author(s) -
Spahr Chris S.,
Susin Santos A.,
Bures Edward J.,
Robinson John H.,
Davis Michael T.,
McGinley Michael D.,
Kroemer Guido,
Patterson Scott D.
Publication year - 2000
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(20000501)21:9<1635::aid-elps1635>3.0.co;2-1
Subject(s) - chemistry , peptide , tandem mass spectrometry , chromatography , mass spectrometry , biotinylation , fragmentation (computing) , protein mass spectrometry , isobaric labeling , bottom up proteomics , top down proteomics , database search engine , peptide mass fingerprinting , proteomics , biochemistry , biology , search engine , ecology , information retrieval , computer science , gene
A rapid means of identifying many components in an enriched mixture of proteins is enzymatic digestion of the entire protein fraction. This complex peptide mixture is then subjected to reversed‐phase high performance liquid chromatography (HPLC) coupled on‐line with a mass spectrometer capable of data‐dependent ion selection for fragmentation (LC‐tandem mass spectrometry; MS/MS). Thus, as many peptides as possible in the sample are fragmented to produce MS/MS spectra, which can then be searched against sequence databases. Ideally, one peptide from each protein in the mixture would be fragmented and identified. To this end, we employed an affinity selection method to capture cysteinyl peptides and thereby simplify the mixture. Both the captured cysteinyl and the noncysteinyl peptides are analyzed by LC‐MS/MS, to increase the numer of proteins identified. The method was tested on a limited set of standard proteins and applied to the analysis of a protein fraction obtained from isolated mitochondria treated with atractyloside. To further increase the number of different precursor ions selected for fragmentation, dynamic exclusion and ion selection from multiple narrow mass ranges of consecutive runs were employed.