The effect of post-translational and process-induced modifications on the multistage gas-phase fragmentation reactions of protonated peptides.

Significant effort has been extended in recent years toward the development and application of 'targeted' approaches for the identification, characterization and quantitative analysis of post-translational or process-induced protein modifications, based on the multistage tandem mass spectrometry (MS/MS and MS(3)) fragmentation reactions of their proteolytically derived peptide ions. Although these approaches have been successfully employed to date, the development of an improved understanding of the mechanisms and other factors (e.g.

Experimental approach for deep proteome measurements from small-scale microbial biomass samples.

Many methods of microbial proteome characterizations require large quantities of cellular biomass (>1-2 g) for sample preparation and protein identification. Our experimental approach differs from traditional techniques by providing the ability to identify the proteomic state of a microbe from a few milligrams of starting cellular material. The small-scale, guanidine lysis method minimizes sample loss by achieving cellular lysis and protein digestion in a single-tube experiment.

Automated neutral loss and data dependent energy resolved "pseudo MS3" for the targeted identification, characterization and quantitative analysis of methionine- containing peptides.

A strategy involving the fixed-charge sulfonium ion derivatization, stable isotope labeling, capillary high- performance liquid chromatography and automated data dependent neutral loss scan mode tandem mass spectrometry (MS/MS) and "pseudo multiple mass spectrometry (MS3)" product ion scans in a triple quadrupole mass spectrometer has been developed for the "targeted" gas-phase identification, characterization and quantitative analysis of low abundance methionine-containing peptides present within complex protein digests. Selective gas-phase "enrichment" and identification is performed via neutral loss scan mode MS/MS, by low energy collision-induced dissociation of the derivatized methionine side chain, resulting in the formation of a single characteristic product ion. Structural characterization of identified peptides is then achieved by automatically subjecting the characteristic neutral loss product ion to further dissociation by data dependent product ion scan mode pseudo MS3 under higher collision energy conditions.

The origin and control of ex vivo oxidative peptide modifications prior to mass spectrometry analysis.

The origin and control of ex vivo sample handling related oxidative modifications of methionine-, S-alkyl cysteine-, and tryptophan-containing peptides obtained from typical "in-solution" or "in-gel" proteolytic digestion strategies, have been examined by capillary HPLC and MS/MS. The origin of increased oxidation levels were found to be predominantly associated with the extensive ex vivo sample handling steps required for gel electrophoresis and/or in-gel proteolytic digestion of proteins prior to analysis by MS. Conditions for deliberately controlling the oxidation state (both oxidation and reduction) of these peptides, as well as for those containing cysteine, have been evaluated using a series of model synthetic peptides and standard tryptic protein digests.

Mechanisms for the proton mobility-dependent gas-phase fragmentation reactions of S-alkyl cysteine sulfoxide-containing peptide ions.

Mechanisms for the gas-phase fragmentation reactions of singly and multiply protonated precursor ions of the model S-alkyl cysteine sulfoxide-containing peptides GAILCGAILK, GAILCGAILR, and VTMGHFCNFGK prepared by reaction with iodomethane, iodoacetamide, iodoacetic acid, acrylamide, or 4-vinylpyridine, followed by oxidation with hydrogen peroxide, as well as peptides obtained from an S-carboxyamidomethylated and oxidized tryptic digest of bovine serum albumin, have been examined using multistage tandem mass spectrometry, hydrogen/deuterium exchange and molecular orbital calculations (at the B3LYP/6-31 + G(d,p) level of theory). Consistent with previous reports, CID-MS/MS of the S-alkyl cysteine sulfoxide-containing peptide ions resulted in the dominant "non-sequence" neutral loss of an alkyl sulfenic acid (XSOH) from the modified cysteine side chains under conditions of low proton mobility, irrespective of the alkylating reagent employed. Dissociation of uniformly deuterated precursor ions of these model peptides determined that the loss of alkyl sulfenic acid in each case occurred via a "charge-remote" five-centered cis-1,2 elimination reaction to yield a dehydroalanine-containing product ion.