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Application of capillary isotachophoresis‐based multidimensional separations coupled with electrospray ionization‐tandem mass spectrometry for characterization of mouse brain mitochondrial proteome
Author(s) -
Fang Xueping,
Wang Weijie,
Yang Li,
Chandrasekaran Krish,
Kristian Tibor,
Balgley Brian M.,
Lee Cheng S.
Publication year - 2008
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/elps.200700609
Subject(s) - proteome , peptide , tandem mass spectrometry , chemistry , proteomics , mass spectrometry , peptide mass fingerprinting , chromatography , electrospray ionization , mitochondrion , isobaric labeling , computational biology , biochemistry , biology , protein mass spectrometry , gene
By employing a capillary ITP (CITP)/CZE‐based proteomic technology, a total of 1795 distinct mouse Swiss‐Prot protein entries (or 1705 nonredundant proteins) are identified from synaptic mitochondria isolated from mouse brain. The ultrahigh resolving power of CITP/CZE is evidenced by the large number of distinct peptide identifications measured from each CITP fraction together with the low peptide fraction overlapping among identified peptides. The degree of peptide overlapping among CITP fractions is even lower than that achieved using combined CIEF/nano‐RP LC separations for the analysis of the same mitochondrial sample. When evaluating the protein sequence coverage by the number of distinct peptides mapping to each mitochondrial protein identification, CITP/CZE similarly achieves superior performance with 1041 proteins (58%) having 3 or more distinct peptides, 233 (13%) having 2 distinct peptides, and 521 (29%) having a single distinct peptide. The reproducibility of protein identifications is found to be around 86% by comparing proteins identified from repeated runs of the same mitochondrial sample. The analysis of the mouse mitochondrial proteome by two CITP/CZE runs results in the detection of 2095 distinct mouse Swiss‐Prot protein entries (or 1992 nonredundant proteins), corresponding to 59% coverage of the updated Maestro mitochondrial reference set. The collective analysis from combined CITP/CZE and CIEF‐based proteomic studies yields the identification of 2191 distinct mitochondrial protein entries (or 2082 nonredundant proteins), corresponding to 76% coverage of the MitoP2‐database reference set.