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Site-specific glycosylation profile of influenza A (H1N1) hemagglutinin through tandem mass spectrometry.

ABSTRACT: The study of influenza virus evolution in humans has revealed a significant role of glycosylation profile alterations in the viral glycoproteins - hemagglutinin (HA) and neuraminidase (NA), in the emergence of both seasonal and pandemic strains. Viral antigenic drift can modify the number and location of glycosylation sites, altering a wide range of biological activities and the antigenic properties of the strain. In view of the key role of glycans in determining antigenicity, elucidating the glycosylation profiles of influenza strains is a requirement towards the development of improved vaccines. Sequence-based analysis of viral RNA has provided great insight into the role of glycosite modifications in altering virulence and pathogenicity. Nonetheless, this sequence-based approach can only predict potential glycosylation sites. Due to experimental challenges, experimental confirmation of the occupation of predicted glycosylation sites has only been carried out for a few strains. Herein, we utilized HCD/CID-MS/MS tandem mass spectrometry to characterize the site-specific profile of HA of an egg-grown H1N1 reference strain (A/New Caledonia/20/1999). We confirmed experimentally the occupancy of glycosylation sites identified by primary sequence analysis and determined the heterogeneity of glycan structures. Four glycosylation sequons on the stalk region (N28, N40, N304 and N498) and four on the globular head (N71, N104, N142 and N177) of the protein are occupied. Our results revealed a broad glycan microheterogeneity, i.e., a great diversity of glycan compositions present on each glycosite. The present methodology can be applied to characterize other viruses, particularly different influenza strains, to better understand the impact of glycosylation on biological activities and aid the improvement of influenza vaccines.

SUBMITTER: Cruz E

PROVIDER: S-EPMC5861804 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Site-specific glycosylation profile of influenza A (H1N1) hemagglutinin through tandem mass spectrometry.

Cruz Esteban E Cain Joel J Crossett Ben B Kayser Veysel V

Human vaccines & immunotherapeutics 20171117 3

The study of influenza virus evolution in humans has revealed a significant role of glycosylation profile alterations in the viral glycoproteins - hemagglutinin (HA) and neuraminidase (NA), in the emergence of both seasonal and pandemic strains. Viral antigenic drift can modify the number and location of glycosylation sites, altering a wide range of biological activities and the antigenic properties of the strain. In view of the key role of glycans in determining antigenicity, elucidating the gl ...[more]

PMID: 29048990

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Project description:Glycosylation is a common protein modification with a significant role in many vital cellular processes and human diseases, making the characterization of protein-attached glycan structures important for understanding cell biology and disease processes. Direct analysis of protein N-glycosylation by tandem mass spectrometry of glycopeptides promises site-specific elucidation of N-glycan microheterogeneity, something that detached N-glycan and deglycosylated peptide analyses cannot provide. However, successful implementation of direct N-glycopeptide analysis by tandem mass spectrometry remains a challenge. In this work, we consider algorithmic techniques for the analysis of LC-MS/MS data acquired from glycopeptide-enriched fractions of enzymatic digests of purified proteins. We implement a computational strategy that takes advantage of the properties of CID fragmentation spectra of N-glycopeptides, matching the MS/MS spectra to peptide-glycan pairs from protein sequences and glycan structure databases. Significantly, we also propose a novel false discovery rate estimation technique to estimate and manage the number of false identifications. We use a human glycoprotein standard, haptoglobin, digested with trypsin and GluC, enriched for glycopeptides using HILIC chromatography, and analyzed by LC-MS/MS to demonstrate our algorithmic strategy and evaluate its performance. Our software, GlycoPeptideSearch (GPS), assigned glycopeptide identifications to 246 of the spectra at a false discovery rate of 5.58%, identifying 42 distinct haptoglobin peptide-glycan pairs at each of the four haptoglobin N-linked glycosylation sites. We further demonstrate the effectiveness of this approach by analyzing plasma-derived haptoglobin, identifying 136 N-linked glycopeptide spectra at a false discovery rate of 0.4%, representing 15 distinct glycopeptides on at least three of the four N-linked glycosylation sites. The software, GlycoPeptideSearch, is available for download from http://edwardslab.bmcb.georgetown.edu/GPS .

Dataset Information

Site-specific glycosylation profile of influenza A (H1N1) hemagglutinin through tandem mass spectrometry.

Publications

Site-specific glycosylation profile of influenza A (H1N1) hemagglutinin through tandem mass spectrometry.

Similar Datasets

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