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Glycan side reaction may compromise ETD-based glycopeptide identification.

ABSTRACT: Tris(hydroxymethyl)aminomethane (Tris) is one of the most frequently used buffer ingredients. Among other things, it is recommended and is usually used for lectin-based affinity enrichment of glycopeptides. Here we report that sialic acid, a common 'capping' unit in both N- and O-linked glycans may react with this chemical, and this side reaction may compromise glycopeptide identification when ETD spectra are the only MS/MS data used in the database search. We show that the modification may alter N- as well as O-linked glycans, the Tris-derivative is still prone to fragmentation both in 'beam-type' CID (HCD) and ETD experiments, at the same time--since the acidic carboxyl group was 'neutralized'--it will display a different retention time than its unmodified counterpart. We also suggest solutions that--when incorporated into existing search engines--may significantly improve the reliability of glycopeptide assignments.

SUBMITTER: Darula Z

PROVIDER: S-EPMC4036456 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Glycan side reaction may compromise ETD-based glycopeptide identification.

Darula Zsuzsanna Z Medzihradszky Katalin F KF

Journal of the American Society for Mass Spectrometry 20140325 6

Tris(hydroxymethyl)aminomethane (Tris) is one of the most frequently used buffer ingredients. Among other things, it is recommended and is usually used for lectin-based affinity enrichment of glycopeptides. Here we report that sialic acid, a common 'capping' unit in both N- and O-linked glycans may react with this chemical, and this side reaction may compromise glycopeptide identification when ETD spectra are the only MS/MS data used in the database search. We show that the modification may alte ...[more]

PMID: 24664807

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Project description:We identified a novel pathogenicity island in Staphylococcus aureus which contains open reading frames (ORFs) similar to the exfoliative toxin (ET) gene, glutamyl endopeptidase gene, and edin-B gene in tandem and the phage resistance gene, flanked by hsdM, hsdS (restriction and modification system), and IS256. The protein encoded by the ET-like gene showed 40, 59, and 68% amino acid sequence identities with exfoliative toxin A (ETA), exfoliative toxin B (ETB), and Staphylococcus hyicus ETB (ShETB), respectively. When injected into neonatal mice, the recombinant protein derived from the ET-like gene induced exfoliation of the skin with loss of cell-to-cell adhesion in the upper part of the epidermis as observed in histological examinations, just as was found in neonatal mice injected with ETA or ETB. Western blot analysis indicated that the recombinant protein is serologically distinct from ETA and ETB. Therefore, the product encoded by this new ORF is a new ET member produced by S. aureus and is termed ETD. ETD did not induce blisters in 1-day-old chickens. In the skins of mice injected with ETD, cell surface staining of desmoglein 1 (Dsg1), a cadherin type cell-to-cell adhesion molecule in desmosomes, was abolished without affecting that of desmoglein 3 (Dsg3). Furthermore, in vitro incubation of the recombinant extracellular domains of Dsg1 and Dsg3 with the recombinant protein demonstrated that both mouse and human Dsg1, but not Dsg3, were directly cleaved in a dose-dependent manner. These results demonstrate that ETD and ETA induce blister formation by identical pathophysiological mechanisms. Clinical strains positive for edin-B were suggested to be clonally associated, and all edin-B-positive strains tested were positive for etd. Among 18 etd-positive strains, 12 produced ETD extracellularly. Interestingly, these strains are mainly isolated from other sources of infections and not from patients with bullous impetigo or staphylococcal scalded-skin syndrome. This strongly suggests that ETD might play a pathogenic role in a broader spectrum of bacterial infections than previously considered.

Project description:Six pairs of transcription profiles between glycopeptide-intermediate S. aureus (GISA [or vancomycin-intermediate S. aureus; VISA]) and glycopeptide-susceptible S. aureus (vancomycin-susceptible S. aureus [VSSA], including glycopeptide-susceptible isogenic mutants from VISA) strains were compared using a microarray. Ninety-two open reading frames which were or tended to be increased in transcription in VISA in at least five out of six array combination pairs were evaluated for their effects on glycopeptide susceptibility by introducing these genes one by one into VSSA strain N315 to construct an overexpression library. By screening the library, 17 genes including 8 novel genes were identified as associated with glycopeptide resistance since their experimental overexpression reduced vancomycin and/or teicoplanin susceptibility of N315. The raised MICs of vancomycin and teicoplanin were 1.25 to 3.0 and 1.5 to 6.0 mg/liter, respectively, as compared to 1.0 mg/liter of N315. Three of these genes, namely graF, msrA2, and mgrA, also raised the oxacillin MIC from 8.0 mg/liter for N315 to 64 to approximately 128 mg/liter when they were overexpressed in N315. Their contribution to vancomycin and beta-lactam resistance was further supported by gene knockout and trans-complementation assay. By using a plasmid-based promoter-green fluorescent protein gene (gfp) transcriptional fusion system, graF promoter-activated cells were purified, and subsequent susceptibility tests and Northern blot analysis demonstrated that the cells with up-regulated activity of graF promoter showed reduced susceptibility to vancomycin, teicoplanin, and oxacillin. In addition, cell morphology studies showed that graF and msrA2 overexpression increased cell wall thickness of N315 by factors of 23.91 and 22.27%, respectively, accompanied by glycopeptide MIC increments of 3- to 6-fold, when they were overexpressed in N315. Moreover, extended experiments and analyses indicate that many of the genes identified above are related to the cell wall biosynthetic pathway, including active nutrient transport systems. We propose that the genes which raise glycopeptide resistance in S. aureus function toward altering the cell wall metabolic pathway.

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Glycan side reaction may compromise ETD-based glycopeptide identification.

Publications

Glycan side reaction may compromise ETD-based glycopeptide identification.

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