title: Application of capillary isotachophoresis-based multidimensional separations coupled with electrospray ionization-tandem mass spectrometry for characterization of mouse brain mitochondrial proteome.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
authors.forename: Xueping
authors.initials: X
authors.lastname: Fang
authors.forename: Weijie
authors.initials: W
authors.lastname: Wang
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.forename: Krish
authors.initials: K
authors.lastname: Chandrasekaran
authors.forename: Tibor
authors.initials: T
authors.lastname: Kristian
authors.forename: Brian M
authors.initials: BM
authors.lastname: Balgley
authors.forename: Cheng S
authors.initials: CS
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002458:Cell Fractionation; D019075:Electrophoresis, Capillary / Q000379:methods*; D051379:Mice; D008928:Mitochondria; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010085:Oxidative Phosphorylation; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013574:Synaptosomes / Q000737:chemistry
chemical_list: D024101:Mitochondrial Proteins; D020543:Proteome