Utility of CE−MS Data in Protein Identification

Abstract

A new method for displaying CE-MALDI-MS data for proteolytic digests is described. This data display mode yields distinct charge-based trends for plots of mass-to-charge (m/z) versus CE migration time. These trends arise owing to the in-solution charge state of the peptides, and this interpretation was confirmed by using empirical peptide electromigration models and peptide standards as charge-state markers. These charge-state specific trends exhibit analytical utility by providing additional chemical information about the peptides, which increases the confidence level of peptide identification and provides a rapid means for screening for posttranslationally modified peptides.

Naturally Occurring Human Urinary Peptides for Use in Diagnosis of Chronic Kidney Disease

“…For further validation of obtained peptide identifications, the strict correlation between peptide charge at pH 2 and CE migration time was utilized to minimize false positive identification rates (42,49). As depicted in Fig.…”

“…Specifically, the peptides in the right line contain no basic amino acids; only the N terminus of the peptide is positively charged at pH 2. In contrast, peptides in the other lines (from right to left) show increasing numbers of basic amino acids in addition to their N-terminal ammonium group (49). The calculated CE migration time of the sequence candidate based on its peptide sequence (number of basic amino acids) was compared with the experimental migration time.…”

“…Sequence information was obtained for 444 of the 5,010 peptides constituting the human urinary peptidome database. 3 As described previously (42,49), the migration time in CE depends on peptide mass and charge state at pH 2, equaling the number of free amino groups (N-terminal and basic amino acids). Therefore, it is not a prerequisite to use CE separation for MS/MS sequencing as the number of basic amino acids as well as the calculated mass serve to assign a putative sequence to a signal in the CE-MS spectrum.…”

CE‐TOF/MS: Fundamental concepts, instrumental considerations and applications

“…Numerous software have been developed to extract information from these highly complex spectra [99]. In the domain of data treatment, Williams et al [100] recently described a method for displaying CE-MALDI-TOF/MS data of proteolytic digests. These data display mode yields distinct chargebased trends for plots of m/z versus CE migration time.…”

Recent applications of capillary electrophoresis–mass spectrometry (CE–MS): CE performing functions beyond separation

“…5). In another example, a map of log( eff ) vs. log(m/z) was found to exhibit charge-state specific trends, which was useful in providing additional information on the peptides and in the screening for post-translationally modified peptides [152].…”