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., proton mobility, peptide conformation, product ion structures, etc.) that influence the multistage fragmentation reactions of modified peptide ions would facilitate further advances in the field. In this review, the important role of such mechanistic studies for rationalizing the effect of post-translational (e.g., phosphoserine- and phosphothreonine-containing peptides) and process-induced (e.g., oxidative modifications of methionine- and S-alkyl cysteine-containing peptides) protein modifications on the multistage collision induced dissociation gas-phase fragmentation reactions of proteolytically derived peptide ions are highlighted. Furthermore, recent efforts toward the development of chemical derivatization strategies for controlling and directing the gas-phase fragmentation reactions of protonated peptides toward the formation of analytically useful fragmentation pathways will be discussed, as well as the use of alternative dissociation techniques including electron capture dissociation (ECD) and electron transfer dissociation (ETD).

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. For this experimental approach, the freshwater microbe Shewanella oneidensis MR-1 and the purple non-sulfur bacterium Rhodopseudomonas palustris CGA0010 were used as model organisms for technology development and evaluation. A 2-D LC-MS/MS comparison between a standard sonication lysis method and the small-scale guanidine lysis techniques demonstrates that the guanidine lysis method is more efficient with smaller sample amounts of cell pellet (i.e., down to 1 mg). The described methodology enables deeper proteome measurements from a few milliliters of confluent bacterial cultures. We also report a new protocol for efficient lysis from small amounts of natural biofilm samples for deep proteome measurements, which should greatly enhance the emerging field of environmental microbial community proteomics. This straightforward sample boiling protocol is complementary to the small-scale guanidine lysis technique, is amenable for small sample quantities, and requires no special reagents that might complicate the MS measurements.

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. Quantitative analysis is achieved by measurement of the abundances of characteristic product ions formed by sequential neutral loss scan mode MS/MS experiments from "light" (12C) and "heavy" (13C) stable isotope encoded fixed-charge derivatized peptides. In contrast to MS-based quantitative analysis strategies, the neutral loss scan mode MS/MS method employed here was able to achieve accurate quantification for individual peptides at levels as low as 100 fmol and at abundance ratios ranging from 0.1 to 10, present within a complex protein digest.

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. Essentially complete oxidation of methionine- and S-alkyl cysteine-containing peptides was achieved by reaction with 30% hydrogen peroxide/5% acetic acid at room temperature for 30 min. Under these conditions, cysteine was also converted to cysteic acid, while only limited oxidation of tryptophan to oxindolylalanine, and methionine and S-alkyl cysteine sulfoxides to their respective sulfones, were observed. Efficient reduction of methionine- and S-alkyl cysteine sulfoxide-containing peptides was achieved by reaction in 1 M dimethylsulfide/10 M hydrochloric acid at room temperature for 10 and 45 min, respectively. None of the reduction conditions evaluated were found to result in the reduction of oxindolylalanine, cysteic acid, or methionine sulfone.

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. Similarly, the charge state dependence to the mechanisms and product ion structures for the losses of CO(2), CO(2) + H(2)O and CO(2) + CH(2)O from S-carboxymethyl cysteine sulfoxide-containing peptides, and for the losses of CH(2)CHCONH(2) and CH(2)CHC(5)H(4)N, respectively, from S-amidoethyl and S-pyridylethyl cysteine sulfoxide-containing peptide ions have also been determined. The results from these studies indicate that both the proton mobility of the peptide precursor ion and the nature of the S-alkyl substituent have a significant influence on the abundances and charge states of the product ions resulting from the various competing fragmentation pathways.