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1.
Mol Cell Proteomics ; 19(9): 1561-1574, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32576591

RESUMO

Mass spectrometry has become an indispensable tool for the characterization of glycosylation across biological systems. Our ability to generate rich fragmentation of glycopeptides has dramatically improved over the last decade yet our informatic approaches still lag behind. Although glycoproteomic informatics approaches using glycan databases have attracted considerable attention, database independent approaches have not. This has significantly limited high throughput studies of unusual or atypical glycosylation events such as those observed in bacteria. As such, computational approaches to examine bacterial glycosylation and identify chemically diverse glycans are desperately needed. Here we describe the use of wide-tolerance (up to 2000 Da) open searching as a means to rapidly examine bacterial glycoproteomes. We benchmarked this approach using N-linked glycopeptides of Campylobacter fetus subsp. fetus as well as O-linked glycopeptides of Acinetobacter baumannii and Burkholderia cenocepacia revealing glycopeptides modified with a range of glycans can be readily identified without defining the glycan masses before database searching. Using this approach, we demonstrate how wide tolerance searching can be used to compare glycan use across bacterial species by examining the glycoproteomes of eight Burkholderia species (B. pseudomallei; B. multivorans; B. dolosa; B. humptydooensis; B. ubonensis, B. anthina; B. diffusa; B. pseudomultivorans). Finally, we demonstrate how open searching enables the identification of low frequency glycoforms based on shared modified peptides sequences. Combined, these results show that open searching is a robust computational approach for the determination of glycan diversity within bacterial proteomes.


Assuntos
Proteínas de Bactérias/análise , Glicopeptídeos/análise , Peptídeos/análise , Polissacarídeos/análise , Proteoma/análise , Proteômica/métodos , Acinetobacter baumannii/química , Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/química , Burkholderia/química , Burkholderia/metabolismo , Campylobacter/química , Campylobacter/metabolismo , Cromatografia Líquida , Bases de Dados de Proteínas , Glicopeptídeos/química , Glicosilação , Peptídeos/química , Proteoma/química , Software , Espectrometria de Massas em Tandem
2.
J Proteome Res ; 20(1): 599-612, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33125241

RESUMO

Hydrophilic interaction liquid chromatography (HILIC) glycopeptide enrichment is an indispensable tool for the high-throughput characterization of glycoproteomes. Despite its utility, HILIC enrichment is associated with a number of shortcomings, including requiring large amounts of starting materials, potentially introducing chemical artifacts such as formylation when high concentrations of formic acid are used, and biasing/undersampling specific classes of glycopeptides. Here, we investigate HILIC enrichment-independent approaches for the study of bacterial glycoproteomes. Using three Burkholderia species (Burkholderia cenocepacia, Burkholderia Dolosa, and Burkholderia ubonensis), we demonstrate that short aliphatic O-linked glycopeptides are typically absent from HILIC enrichments, yet are readily identified in whole proteome samples. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS) fractionation, we show that at high compensation voltages (CVs), short aliphatic glycopeptides can be enriched from complex samples, providing an alternative means to identify glycopeptide recalcitrant to hydrophilic-based enrichment. Combining whole proteome and FAIMS analyses, we show that the observable glycoproteome of these Burkholderia species is at least 25% larger than what was initially thought. Excitingly, the ability to enrich glycopeptides using FAIMS appears generally applicable, with the N-linked glycopeptides of Campylobacter fetus subsp. fetus also being enrichable at high FAIMS CVs. Taken together, these results demonstrate that FAIMS provides an alternative means to access glycopeptides and is a valuable tool for glycoproteomic analysis.


Assuntos
Burkholderia , Glicopeptídeos , Espectrometria de Mobilidade Iônica , Proteoma , Burkholderia/metabolismo , Campylobacter , Interações Hidrofóbicas e Hidrofílicas
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