Large-scale intact glycopeptide identification by Mascot database search

Abstract

Workflows capable of determining glycopeptides in large-scale are missing in the field of glycoproteomics. We present an approach for automated annotation of intact glycopeptide mass spectra. The steps in adopting the Mascot search engine for intact glycopeptide analysis included: (i) assigning one letter codes for monosaccharides, (ii) linearizing glycan sequences and (iii) preparing custom glycoprotein databases. Automated annotation of both N- and O-linked glycopeptides was proven using standard glycoproteins. In a large-scale study, a total of 257 glycoproteins containing 970 unique glycosylation sites and 3447 non-redundant N-linked glycopeptide variants were identified in 24 serum samples. Thus, a single tool was developed that collectively allows the (i) elucidation of N- and O-linked glycopeptide spectra, (ii) matching glycopeptides to known protein sequences, and (iii) high-throughput, batch-wise analysis of large-scale glycoproteomics data sets.

Urinary Glycopeptide Analysis for the Investigation of Novel Biomarkers

“…Recent advancements in glycoproteomics for the characterization of glycoproteins open research avenues toward better understanding of their (patho)physiological role. Increased attention has been given to glycoprotein/glycopeptide composition, determining the glycosylation form, glycan structure, and attachment sites …”

Capturing site-specific heterogeneity with large-scale N-glycoproteome analysis

“…As such, multiple dissociation strategies (mainly electron-driven dissociation and collision-based methods) are increasingly used to access both glycan and peptide information from intact glycopeptides. 11,12,[21][22][23][24][25][26][13][14][15][16][17][18][19][20] Even with these methods, large-scale analysis of intact glycopeptides remains largely limited to fewer than ~1,000 unique glycosite identifications from any one system or tissue (approximately an order of magnitude behind other PTMs), [27][28][29][30][31][32] and few studies assess heterogeneity across the glycoproteome with site-specific resolution.…”

“…As such, multiple dissociation strategies (mainly electron-driven dissociation and collision-based methods) are increasingly used to access both glycan and peptide information from intact glycopeptides. 11,12,[21][22][23][24][25][26][13][14][15][16][17][18][19][20] Even with these methods, large-scale analysis of intact glycopeptides remains largely limited to fewer than ~1,000 unique glycosite identifications from any one system or tissue (approximately an order of magnitude behind other PTMs), [27][28][29][30][31][32] and few studies assess heterogeneity across the glycoproteome with site-specific resolution.Activated-ion electron transfer dissociation (AI-ETD) has rapidly developed as a highly effective tandem MS approach for proteomic applications. [33][34][35][36][37][38][39][40][41] The method uses concurrent infrared (IR) photoactivation to improve dissociation efficiencies and increase product ion generation in electron transfer dissociation (ETD) reactions.…”

Multi-isotype Glycoproteomic Characterization of Serum Antibody Heavy Chains Reveals Isotype- and Subclass-Specific N-Glycosylation Profiles

“…Glycoproteomic-based methods for the analysis of immunoglobulins, primarily IgG derived both from serum and monoclonal sources, have contributed greatly to the understanding of the impact of N-glycan heterogeneity on the activity of these important molecules (25, 39 -43). Recently, collision-induced dissociation (CID) with elevated dissociation energy (44) and higher-energy collisional dissociation (HCD) with stepped collision energy (45,46) have shown great promise for glycopeptide analysis. Using the latter approach, a glycopeptide precursor ion is fragmented using two or more collision energies, and the product ions from these independ- 1 The abbreviations used are: ADCC, antibody-dependent cellular cytotoxicity; ACD, acid citrate dextrose solution; AGC, automatic gain control; ADCP, antibody-dependent cellular phagocytosis; ADNP, antibody-dependent neutrophil phagocytosis; ADCD, antibody-dependent and complement deposition; APTS, 8-aminopyrene-1,3,6trisulfonic acid; CE, Capillary electrophoresis; DHB, 2,5-dihydroxybenzoic acid; DTT, dithiothreitol; EIC, extracted ion chromatogram; ER, endoplasmic reticulum; Fc, fragment crystallizable; FcR, fc-receptor; IgA, immunoglobulin A; IgE, immunoglobulin E; IgG, immunoglobulin G; IgM, immunoglobulin M; NCE, normalized collision energy; nLC-MS/MS, nano-liquid chromatography-tandem mass spectrometry; PNGase F, peptide N-glycosidase F.…”