Affinity Enrichment and Characterization of Mucin Core-1 Type Glycopeptides from Bovine Serum

Glycosylation is one of the most common post-translational modifications of proteins. It is estimated that over half of mammalian proteins are glycosylated. Patients with several autoimmune disorders, chronic inflammatory diseases, and some infectious diseases exhibit abnormal glycosylation of serum immunoglobulins and other glycoproteins (1-5). The biological functions of these modifications in health and disease have become a significant area of interest in biomedical research (6). A subset of these glycoproteins has clustered sites of O-glycosylation with serine-and threonine-rich stretches within the amino acid sequence. Mucins, such as membrane-associated MUC1, are perhaps the best known family of proteins that are heavily O-glycosylated. Their altered expression and aberrant glycosylation have made them potential targets as biomarkers for early detection of cancer (7). Immunoglobulin A1 (IgA1) 1 contains both O-and N-glycans (Fig. 1). Aberrant O-glycosylation of IgA1 is involved in the pathogenesis of IgA nephropathy (IgAN) and the closely related Henoch-Schö nlein purpura nephritis (1, 8). Interestingly, the aberrantly glycosylated molecules, IgA1 in IgAN and MUC1 in cancer, are recognized by the immune system as neoepitopes as evidenced by formation of specific antibodies (9 -11). Mucin-like bacterial surface proteins exhibit similar properties: the molecules have clustered bacterial O-glycans that mediate cellular adhesion, and blocking antibodies target these glycan-containing epitopes (12).An O-glycosylated protein from a single source contains a population of variably O-glycosylated isoforms that show a distinct distribution of microheterogeneity of the O-glycan chains in terms of number, sites of attachment, and composition. Characterizing these clustered sites and understanding how the distributions change under different biological conditions or disease states are an analytical challenge. Enzymatic or chemical release of O-glycans is not selective. The heterogeneity, composition, and quantitative aspects of different O-glycan chains can be assessed and quantified by gas chromatographic and/or mass spectrometric techniques.

Section: Application Of Ecd/etd To Clustered Sites Of O-glycosylationsupporting

confidence: 90%

“…Broader studies on peptide and glycopeptide ETD fragmentation have indicated that peptide charge density (residues per charge) plays a significant role in the amount of backbone fragmentation (45,46). The difference in observed fragments for the 3 ϩ and 4…”

Section: Application Of Ecd/etd To Clustered Sites Of O-glycosylationmentioning

confidence: 99%

Clustered O-Glycans of IgA1

Takahashi

Wall

Suzuki

et al. 2010

“…Additionally, the exact glycosylation site of identified peptides containing several Ser/Thr residues cannot be predicted due to the lack of a consensus sequence for mucin-type O-glycosylation. The alternative fragmentation techniques electron capture dissociation (ECD) (38,39) and electron transfer dissociation (ETD) (40) have been introduced for site-specific analysis of CID-labile PTMs but characterization of protein O-glycosylations using ECD/ETD have generally been limited to synthetic glycopeptides or single glycoproteins (41)(42)(43)(44)(45) (46). We have previously developed a sialic acid specific capture-and-release protocol for the enrichment of both N-and O-glycosylated peptides from sialylated glycoproteins in biological samples using hydrazide chemistry (37).…”

mentioning

confidence: 99%

Human Urinary Glycoproteomics; Attachment Site Specific Analysis of N- and O-Linked Glycosylations by CID and ECD

Halim

Nilsson

Rüetschi

et al. 2012

142

151

“…Furthermore, even HCD-MS/MS begins to lose its usefulness when the number of saccharide residues exceeds ϳ4 -5 (whether in a single glycan or spread out among multiple sites), reverting again to domination by sugar losses and lack of peptide fragmentation at reasonable collision energies (5,12). This situation is alleviated partly by the use of electron capture dissociation/ETD-MS/MS, which provides peptide sequence ions with substantial retention of the glycan, making its site known in a relatively uncomplicated way.…”

mentioning

confidence: 99%

“…However, even electron capture dissociation/ETD-MS/MS has its limitations, with heavy glycosylation and/or a lack of abundant precursors with required multiple charge states (e.g. ϩ3, ϩ4, and ϩ5) being two typical reasons for failure to observe sufficient fragmentation for site-specific sequence analysis (5,12,13). The limitations can become acute when there are many adjacent Ser and Thr residues with potential for glycosylation and many Pro residues interrupting the continuity of c/z fragmentation, both situations being frequent in O-glycosylated peptides.…”

mentioning

confidence: 99%

Enhanced Mass Spectrometric Mapping of the Human GalNAc-type O-Glycoproteome with SimpleCells

Vakhrushev

Steentoft

Vester-Christensen

et al. 2013

100

109

Characterizing protein GalNAc-type O-glycosylation has long been a major challenge, and as a result, our understanding of this glycoproteome is particularly poor. Recently, we presented a novel strategy for high throughput identification of O-GalNAc glycosites using zinc finger nuclease gene-engineered "SimpleCell" lines producing homogeneous truncated O-glycosylation. Total lysates of cells were trypsinized and subjected to lectin affinity chromatography enrichment, followed by identification of GalNAc O-glycopeptides by nLC-MS/MS, with electron transfer dissociation employed to specify sites of O-glycosylation. Here, we demonstrate a substantial improvement in the SimpleCell strategy by including an additional stage of lectin affinity chromatography on secreted glycoproteins from culture media (secretome) and by incorporating pre-fractionation of affinity-enriched glycopeptides via IEF before nLC-MS/MS. We applied these improvements to three human SimpleCells studied previously, and each yielded a substantial increase in the number of O-glycoproteins and O-glycosites identified. We found that analysis of the secretome was an important independent factor for increasing identifications, suggesting that further substantial improvements can also be sought through analysis of subcellular organelle fractions. In addition, we uncovered a substantial nonoverlapping set of O-glycoproteins and O-glycosites using an alternative protease digestion (chymotrypsin). In total, the improvements led to identification of 259 glycoproteins, of which 152 (59%) were novel compared with our previous strategy using the same three cell lines. With respect to individual glycosites, we identified a total of 856 sites, of which 508 (59%) were novel compared with our previous strategy; this includes four new identifications of OGalNAc attached to tyrosine. Furthermore, we uncovered ϳ220 O-glycosites wherein the peptides were clearly identified, but the glycosites could not be unambiguously assigned to specific positions. The improved strategy should greatly facilitate high throughput characterization of the human GalNAc-type O-glycoproteome as well as be applicable to analysis of other O-glycoproteomes.