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Development of serum glycoproteomic profiling technique; simultaneous identification of glycosylation sites and site-specific quantification of glycan structure changes.

ABSTRACT: Characterization and interpretation of disease-associated alterations of protein glycosylation are the central aims of the emerging glycoproteomics projects, which are expected to lead to more sensitive and specific diagnosis and improve therapeutic outcomes for various diseases. Here we report a new approach to identify carbohydrate-targeting serum biomarkers, termed isotopic glycosidase elution and labeling on lectin-column chromatography (IGEL). This technology is based on glycan structure-specific enrichment of glycopeptides by lectin-column chromatography and site-directed tagging of N-glycosylation sites by (18)O during the elution with N-glycosidase. The combination of IGEL with 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) stable isotope labeling enabled us not only to identify N-glycosylation sites effectively but also to compare glycan structures on each glycosylation site quantitatively in a single LC/MS/MS analysis. We applied this method to eight sera from lung cancer patients and controls, and finally identified 107 glycopeptides in their sera, including A2GL_Asn151, A2GL_Asn290, CD14_Asn132, CO8A_Asn417, C163A_Asn64, TIMP1_Asn30, and TSP1_Asn1049 which showed the significant change of the affinity to Concanavalin A (ConA) lectin between the lung cancer samples and the controls (p < 0.05 and more than twofold change). These screening results were further confirmed by the conventional lectin-column chromatography and immunoblot analysis using additional serum samples. Our novel methodology, which should be valuable for diverse biomarker discoveries, can provide high-throughput and quantitative profiling of glycan structure alterations.

SUBMITTER: Ueda K

PROVIDER: S-EPMC2938115 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Publications

Development of serum glycoproteomic profiling technique; simultaneous identification of glycosylation sites and site-specific quantification of glycan structure changes.

Ueda Koji K Takami Sachiko S Saichi Naomi N Daigo Yataro Y Ishikawa Nobuhisa N Kohno Nobuoki N Katsumata Masaru M Yamane Akio A Ota Minoru M Sato Taka-Aki TA Nakamura Yusuke Y Nakagawa Hidewaki H

Molecular & cellular proteomics : MCP 20100901 9

Characterization and interpretation of disease-associated alterations of protein glycosylation are the central aims of the emerging glycoproteomics projects, which are expected to lead to more sensitive and specific diagnosis and improve therapeutic outcomes for various diseases. Here we report a new approach to identify carbohydrate-targeting serum biomarkers, termed isotopic glycosidase elution and labeling on lectin-column chromatography (IGEL). This technology is based on glycan structure-sp ...[more]

PMID: 20811073

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Project description:Antibodies are critical glycoproteins that bridge the innate and adaptive immune systems to provide protection against infection. The isotype/subclass of the antibody, the co-translational N-glycosylation on the CH2 domain, and the remodeling of the N-linked glycans during passage through the ER and Golgi are the known variables within the Fc domain that program antibody effector function. Through investigations of monoclonal therapeutics, it has been observed that addition or removal of specific monosaccharide residues from antibody N-glycans can influence the potency of antibodies, highlighting the importance of thoroughly characterizing antibody N-glycosylation. Although IgGs usually have a single N-glycosylation site and are well studied, other antibody isotypes, e.g. IgA and IgM, that are the first responders in certain diseases, have two to five sites/monomer of antibody, and little is known about their N-glycosylation. Here we employ a nLC-MS/MS method using stepped-energy higher energy collisional dissociation to characterize the N-glycan repertoire and site occupancy of circulating serum antibodies. We simultaneously determined the site-specific N-linked glycan repertoire for IgG1, IgG4, IgA1, IgA2, and IgM in individual healthy donors. Compared with IgG1, IgG4 displayed a higher relative abundance of G1S1F and a lower relative abundance of G1FB. IgA1 and IgA2 displayed mostly biantennary N-glycans. IgA2 variants with the either serine (S93) or proline (P93) were detected. In digests of the sera from a subset of donors, we detected an unmodified peptide containing a proline residue at position 93; this substitution would strongly disfavor N-glycosylation at N92. IgM sites N46, N209, and N272 displayed mostly complex glycans, whereas sites N279 and N439 displayed higher relative abundances of high-mannose glycoforms. This multi-isotype approach is a crucial step toward developing a platform to define disease-specific N-glycan signatures for different isotypes to help tune antibodies to induce protection. Data are available via ProteomeXchange with identifier PXD010911.

Dataset Information

Development of serum glycoproteomic profiling technique; simultaneous identification of glycosylation sites and site-specific quantification of glycan structure changes.

Publications

Development of serum glycoproteomic profiling technique; simultaneous identification of glycosylation sites and site-specific quantification of glycan structure changes.

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