A major goal within the emerging field of proteomics is the systematic identification, characterization and quantitative analysis of protein expression, protein post-translational modifications (PTMs) and specific functional protein-protein interactions that are involved in the regulation/deregulation of normal cellular function. To date, the application of mass spectrometry (MS) and tandem mass spectrometry (MS/MS) based approaches have achieved remarkable progress with regard to the investigation of biological problems. However, the enormous sample mixture complexity and the presence of various PTMs that regulate cellular processes present formidable challenges for the comprehensive analysis of cellular proteomes. Chemical labeling has been a valuable tool to assist in MS-based proteomics, to achieve the goal such as the improved peptide/protein sequence analysis, the enrichment and characterization of protein PTMs, the quantitation of protein expressions or to probe protein structure and protein-protein interactions. Here, the development of fixed charge chemical labeling strategies for selective control of the multistage gas-phase fragmentation reactions of peptide ions is described. In one study, a novel `fixed charge' sulfonium ion-containing `amine reactive' cross-linking reagent S-methyl 5,5’-thiodipentanoylhydroxysuccinimide was synthesized, characterized, and initially applied to model peptides, in order to develop a `targeted' multistage tandem mass spectrometry based approach for the identification and characterization of protein-protein interactions. Under low energy collision induced dissociation (CID)-MS/MS conditions, peptide ions containing this cross-linker are shown to exclusively fragment via facile cleavage of the bond directly adjacent to the `fixed charge', which was found to be independent of the precursor ion charge state or amino acid composition (i.e., proton mobility). Thus, the cross-linked peptides can be effectively identified from unmodified peptides, and the various types of cross-linked products (i.e., intra, inter, and dead-end) that may be formed from a cross-linking reaction are readily distinguished via recognition of their distinct fragmentation patterns. Another study was focused on the development and application of a peptide derivatization strategy using an amine specific sulfonium ion containing reagent S,S’-dimethylthiobutanoylhydroxysuccinimide ester iodide (DMBNHS) combined with CID-MS/MS and electron transfer dissociation (ETD)-MS/MS for the enhanced characterization and quantitative analysis of protein phosphorylation. The introduction of this fixed charge to phosphopeptides is shown to lead to improved ionization efficiencies, and an increase in the abundance of higher charge state precursor ions following electrospray ionization (ESI). Upon CID-MS/MS, the exclusive neutral loss(es) of dimethylsulfide are observed, without loss of the phosphate group(s). The relative abundances of "light" versus "heavy" neutral loss product ions generated from CID-MS/MS of D6-light and D6-heavy DMBNHS stable isotope labeled phosphopeptides enables their differential quantitative analysis, whereas subsequent ETD-MS/MS of the intact precursor ion(s) allows phosphopeptide sequence identification and phosphorylation site characterization, suggesting the reagent holds great promise for studies of protein post-translational modifications.
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