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.

CE–MS for proteomics: Advances in interface development and application

“…A similar strategy was used by Fang et al for the analysis of human saliva and mouse brain mitochondrial proteome [72,73]. Their strategy consisted of an off-line coupling of an ITP/CZE to nano-RPLC-ESI-MS via a fraction collector.…”

Recent advances in capillary electrophoresis‐based proteomic techniques for biomarker discovery

“…A total of 12 110 fully tryptic peptides were detected, leading to the identification of 1795 distinct mouse SwissProt protein entries (or 1705 non-redundant proteins) from a single CITP/CZE-based proteomic study of synaptic mouse brain mitochondria [53]. The reproducibility of protein identifications was evaluated and was found to be around 86% by comparing proteins identified from repeated runs of the same mitochondrial sample.…”

2‐D t‐ITP/CZE determination of clinical urinary proteins using a microfluidic‐chip capillary electrophoresis device

“…Out of the various techniques hyphenated with microchips, ITP has been shown capable to increase the sample loading capacity in addition to desalting, leading to a high enrichment factor after sample clean-up [31][32][33]. Thus, it has been incorporated in the present design of microfluidic chip-CE (microchip-CE) device for the determination of trace analyte proteins in clinical urine samples with interfering high salt and metabolite concentrations.…”