Campylobacter jejuni is a gastrointestinal pathogen that is able to modify membrane and periplasmic proteins by the N-linked addition of a 7-residue glycan at the strict attachment motif (D/E)XNX(S/T). Strategies for a comprehensive analysis of the targets of glycosylation, however, are hampered by the resistance of the glycan-peptide bond to enzymatic digestion or -elimination and have previously concentrated on soluble glycoproteins compatible with lectin affinity and gel-based approaches. We developed strategies for enriching C. jejuni HB93-13 glycopeptides using zwitterionic hydrophilic interaction chromatography and examined novel fragmentation, including collision-induced dissociation (CID) and higher energy collisional (C-trap) dissociation (HCD) as well as CID/electron transfer dissociation (ETD) mass spectrometry. CID/HCD enabled the identification of glycan structure and peptide backbone, allowing glycopeptide identification, whereas CID/ETD enabled the elucidation of glycosylation sites by maintaining the glycan-peptide linkage. A total of 130 glycopeptides, representing 75 glycosylation sites, were identified from LC-MS/MS using zwitterionic hydrophilic interaction chromatography coupled to CID/HCD and CID/ETD. CID/HCD provided the majority of the identifications (73 sites) compared with ETD (26 sites). We also examined soluble glycoproteins by soybean agglutinin affinity and two-dimensional electrophoresis and identified a further six glycosylation sites. This study more than doubles the number of confirmed N-linked glycosylation sites in C. jejuni and is the first to utilize HCD fragmentation for glycopeptide identification with intact glycan. We also show that hydrophobic integral membrane proteins are significant targets of glycosylation in this organism. Our data demonstrate that peptide-centric approaches coupled to novel mass spectrometric fragmentation techniques may be suitable for application to eukaryotic glycoproteins for simul-
Cysteine (Cys) oxidation is a crucial post-translational modification (PTM) associated with redox signaling and oxidative stress. As Cys is highly reactive to oxidants it forms a range of post-translational modifications, some that are biologically reversible (e.g. disulfides, Cys sulfenic acid) and others (Cys sulfinic [Cys-SO 2 H] and sulfonic [Cys-SO 3 H] acids) that are considered "irreversible." We developed an enrichment method to isolate Cys-SO 2 H/SO 3 H-containing peptides from complex tissue lysates that is compatible with tandem mass spectrometry (MS/MS). The acidity of these post-translational modification (pK a Cys-SO 3 H < 0) creates a unique charge distribution when localized on tryptic peptides at acidic pH that can be utilized for their purification. The method is based on electrostatic repulsion of Cys-SO 2 H/SO 3 H-containing peptides from cationic resins (i.e. "negative" selection) followed by "positive" selection using hydrophilic interaction liquid chromatography. Modification of strong cation exchange protocols decreased the complexity of initial flowthrough fractions by allowing for hydrophobic retention of neutral peptides. Coupling of strong cation exchange and hydrophilic interaction liquid chromatography allowed for increased enrichment of Cys-SO 2 H/SO 3 H (up to 80%) from other modified peptides. We identified 181 Cys-SO 2 H/SO 3 H sites from rat myocardial tissue subjected to physiologically relevant concentrations of H 2 O 2 (<100 M) or to ischemia/reperfusion (I/R) injury via Langendorff perfusion.
Redox regulation is emerging as an important post-translational modification in cell signaling and pathogenesis. Cysteine (Cys) is the most redox active of the commonly coded amino acids and is thus an important target for redox-based modifications. Reactions that oxidize the Cys sulfur atom to low oxidation states (e.g., disulfide) are reversible, while further reactions to higher oxidation states (e.g., sulfonic acid) may be irreversible under biological conditions. Reversible modifications are particularly interesting as they mediate redox signaling and regulation of proteins under physiological conditions and during adaptation to oxidant stress. An enrichment method that relied on rapid and specific alkylation of free Cys, followed by thiol-based reduction and resin capture by thiol-disulfide exchange chemistry was applied to isolate reversibly modified Cys-containing peptides. Chromatographic conditions were optimized to provide increased specificity by removal of noncovalent interactions. The technique was highly efficient, based on near equimolar reactions with the resin, reproducible and linear for peptide elution, as quantified by label-free mass spectrometry. The method was applied to a complex protein lysate generated from rat myocardial tissue and 6559 unique Cys-containing peptides from 2694 proteins were identified. Comparison with the rat database and previous studies showed effective enrichment of proteins modified by S-nitrosylation, disulfide formation, and Cys-sulfenic acid. Analysis of amino acid sequence features indicated a preference for acidic residues and increased hydrophilicity in the regions immediately up- or downstream of the reactive Cys. This technique is ideally suited for the enrichment and profiling of reversible Cys modifications on a proteome-wide scale.
Aims: Cysteine (Cys) is a major target for redox post-translational modifications (PTMs) that occur in response to changes in the cellular redox environment. We describe multiplexed, peptide-based enrichment and quantitative mass spectrometry (MS) applied to globally profile reversible redox Cys PTM in rat hearts during ischemia/ reperfusion (I/R) in the presence or absence of an aminothiol antioxidant, N-2-mercaptopropionylglycine (MPG). Parallel fractionation also allowed identification of irreversibly oxidized Cys peptides (Cys-SO 2 H/SO 3 H). Results: We identified 4505 reversibly oxidized Cys peptides of which 1372 were significantly regulated by ischemia and/or I/R. An additional 219 peptides (247 sites) contained Cys-SO 2 H/Cys-SO 3 H modifications, and these were predominantly identified from hearts subjected to I/R (n = 168 peptides). Parallel reaction monitoring MS (PRM-MS) enabled relative quantitation of 34 irreversibly oxidized Cys peptides. MPG attenuated a large cluster of I/R-associated reversibly oxidized Cys peptides and irreversible Cys oxidation to less than nonischemic controls (n = 24 and 34 peptides, respectively). PRM-MS showed that Cys sites oxidized during ischemia and/or I/R and ''protected'' by MPG were largely mitochondrial, and were associated with antioxidant functions (peroxiredoxins 5 and 6) and metabolic processes, including glycolysis. Metabolomics revealed I/R induced changes in glycolytic intermediates that were reversed in the presence of MPG, which were consistent with irreversible PTM of triose phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), altered GAPDH enzyme activity, and reduced I/R glycolytic payoff as evidenced by adenosine triphosphate and NADH levels. Innovation: Novel enrichment and PRM-MS approaches developed here enabled large-scale relative quantitation of Cys redox sites modified by reversible and irreversible PTM during I/R and antioxidant remediation. Conclusions: Cys sites identified here are targets of reactive oxygen species that can contribute to protein dysfunction and the pathogenesis of I/R. Antioxid. Redox Signal. 34,
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