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-

changes. These were mainly from predicted extracellular matrix and basement membrane proteins that are implicated in cardiac remodeling. Analysis of N-glycans released from myocardial proteins suggest that the observed changes were not due to significant alterations in N-glycan structures. Altered proteins included the collagen-laminin-integrin complexes and collagen assembly enzymes, cadherins, mast cell proteases, proliferation-associated secreted protein acidic and rich in cysteine, and microfibril-associated proteins. The data suggest that cardiac remodeling is initiated earlier during reperfusion than previously hypothesized.

The opportunistic human pathogen Acinetobacter baumannii is a concern to health care systems worldwide because of its persistence in clinical settings and the growing frequency of multiple drug resistant infections. To combat this threat, it is necessary to understand factors associated with disease and environmental persistence of A. baumannii. Recently, it was shown that a single biosynthetic pathway was responsible for the generation of capsule polysaccharide and O-linked protein glycosylation. Because of the requirement of these carbohydrates for virulence and the non-template driven nature of glycan biogenesis we investigated the composition, diversity, and properties of the Acinetobacter glycoproteome. Utilizing global and targeted mass spectrometry methods, we examined 15 strains and found extensive glycan diversity in the O-linked glycoproteome of Acinetobacter. Comparison of the 26 glycoproteins identified revealed that different A. baumannii strains target similar protein substrates, both in characteristics of the sites of O-glycosylation and protein identity. Surprisingly, glycan micro-heterogeneity was also observed within nearly all isolates examined demonstrating glycan heterogeneity is a widespread phenomena in Acinetobacter O-linked glycosylation. By comparing the 11 main glycoforms and over 20 alternative glycoforms characterized within the 15 strains, trends within the glycan utilized for O-linked glycosylation could be observed. These trends reveal Acinetobacter O-linked glycosylation favors short (three to five residue) glycans with limited branching containing negatively charged sugars such as GlcNAc3NAcA4OAc or legionaminic/pseudaminic acid derivatives. These observations suggest that although highly diverse, the capsule/O-linked glycan biosynthetic pathways generate glycans with similar characteristics across all A. baumannii. Molecular & Cellular

No abstract

We present basic workups and quantitative comparisons for two current generation Orbitrap mass spectrometers, the Q Exactive Plus and Orbitrap Fusion Tribrid, which are widely considered two of the highest performing instruments on the market. We assessed the performance of two quantitative methods on both instruments, namely label-free quantitation and stable isotope labeling using isobaric tags, for studying the heat shock response in Escherichia coli. We investigated the recently reported MS3 method on the Fusion instrument and the potential of MS3-based reporter ion isolation Synchronous Precursor Selection (SPS) and its impact on quantitative accuracy. We confirm that the label-free approach offers a more linear response with a wider dynamic range than MS/MS-based isobaric tag quantitation and that the MS3/SPS approach alleviates but does not eliminate dynamic range compression. We observed, however, that the choice of quantitative approach had little impact on the ability to statistically evaluate the E. coli heat shock response. We conclude that in the experimental conditions tested, MS/MS-based reporter ion quantitation provides reliable biological insight despite the issue of compressed dynamic range, an observation that significantly impacts the choice of instrument.

Cardiovascular disease remains the most common cause of death in the developed world and is predicted by the World Health Organization to kill ϳ20 million people worldwide each year until at least 2015. In light of these figures, work on producing superior tools for clinical use in the cardiovascular field is intensive. As proteins are the primary effectors of cellular function, a significant majority of this work focuses on the role of proteins in the cardiovascular system in physiological and pathological states in order to outline both mechanisms and markers of disease. One of the most effective ways to investigate these on a global basis is through proteomic analysis, which allows for broad spectrum screening of cellular protein or peptide complements during cardiovascular pathogenesis. Furthermore, specific technologies are now available to screen animal model or human blood samples for novel, improved markers of chronic disease states, such as atherosclerosis or for earlier indicators of acute myocardial stress, including ischemia/reperfusion injury and heart failure. This review summarizes current literature on the key aspects of proteomics and peptidomics related to clinical cardiovascular science.

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.

No abstract