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. 2017 Jul;409(19):4483-4505.
doi: 10.1007/s00216-017-0406-7. Epub 2017 Jun 6.

Glycomic and glycoproteomic analysis of glycoproteins-a tutorial

Asif Shajahan  1 , Christian Heiss  1 , Mayumi Ishihara  1 , Parastoo Azadi  2
Affiliations

Glycomic and glycoproteomic analysis of glycoproteins-a tutorial

Asif Shajahan et al. Anal Bioanal Chem. 2017 Jul.

Abstract

The structural analysis of glycoproteins is a challenging endeavor and is under steadily increasing demand, but only a very limited number of labs have the expertise required to accomplish this task. This tutorial is aimed at researchers from the fields of molecular biology and biochemistry that have discovered that glycoproteins are important in their biological research and are looking for the tools to elucidate their structure. It provides brief descriptions of the major and most common analytical techniques used in glycomics and glycoproteomics analysis, including explanations of the rationales for individual steps and references to published literature containing the experimental details necessary to carry out the analyses. Glycomics includes the comprehensive study of the structure and function of the glycans expressed in a given cell or organism along with identification of all the genes that encode glycoproteins and glycosyltransferases. Glycoproteomics which is subset of both glycomics and proteomics is the identification and characterization of proteins bearing carbohydrates as posttranslational modification. This tutorial is designed to ease entry into the glycomics and glycoproteomics field for those without prior carbohydrate analysis experience.

Keywords: Glycan analysis; Glycomics; Glycopeptide; Glycoproteomics; Glycosylation site mapping; Mass spectrometry.

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Conflict of interest statement

Asif Shajahan, Christian Heiss, and Parastoo Azadi work at the Analytical Services & Training Laboratory at the Complex Carbohydrate Research Center and perform glycomics and glycoproteomics analysis as collaboration or fee-for-service. Mayumi Ishihara declares that she has no conflict of interest.

Figures

Fig. 1
Fig. 1
General workflows for glycomics and glycoproteomics analysis. Glycomics involves the release of N-linked glycans (using PNGase F, PNGase A, or hydrazinolysis) and O-linked glycans (by reductive alkaline β-elimination or hydrazinolysis) and subsequent derivatization of glycans (by permethylation or reductive amination with chromophores such as 2-AP) and analysis by MALDI and ESI mass spectrometry. Glycoproteomics comprises of determination of glycosylation at glycopeptide through direct LC-MS/MS analysis of intact glycopeptide and identification of site of glycosylation of O-glycans (BEMAD or ETD) and N-glycans (18O-labeling, endo-H digestion, or ETD) by tandem LC-MS/MS
Fig. 2
Fig. 2
Schematic representation showing mechanism of glycan release from proteins of A N-glycans by PNGase F and optional 18O-labeling [22] and of B O-glycans by reductive β-elimination and optional BEMAD [23]
Fig. 3
Fig. 3
Schematic representation showing the masses of each monosaccharide component on a permethylated glycan. A Nonreduced permethylated glycan. B Reduced permethylated glycan
Fig. 4
Fig. 4
Advantage of permethylation of glycan in the differentiation of glycan isomers via CID MSn. The m/z value of sodium adduct of permethylated glycans and native glycans of N-glycan Man5GlcNAc2 (in brackets) which are provided under each oligosaccharide fragments shows that only MSn of permethylated glycans possess key fragments (in bold) that can help to differentiate isomers A and B. The circles superimposing the bonds mean that the oxygen atoms of these glycosidic bonds are included in the fragment, i.e., they are Y-ions (see Fig. 9B) [41]
Fig. 5
Fig. 5
Determination of linkage positions of glycans through permethylation: permethylated glycans are acid hydrolyzed, acetylated, and reduced, and the resulting PMAAs are analyzed by GC-MS. In the PMAAs, the location of acetyl groups indicate linkage positions, whereas methyl groups indicate positions that were unsubstituted in the original glycan. During MS, fragmentation occurs preferentially next to methyl groups. The example here is a Man6GlcNAc2 N-glycan. The symbolism shown in A is explained in Fig. 10. The MS fragmentations are shown in B
Fig. 6
Fig. 6
A An example MS/MS-HCD spectrum of a glycopeptide carrying a high-mannose type N-glycan, Man5GlcNAc2 on asparagine. It showed a series of glycan oxonium ions in the low mass region at m/z 163.0603, 204.0868, 366.1398, and 528.1929, respectively. A series of fragment ions due to neutral loss of the glycan moiety were observed as the main fragment ions in the HCD spectrum. Trace amounts of y-type and b-type peptide fragment ions were detected, confirming the sequence of the peptide backbone. B An example of MS/MS-ETD spectra from a glycopeptide carrying Man5GlcNAc2, showing fragment ions with minimal neutral loss of glycan moiety. All expected peptide c-type and z-type fragment ions were detected except c8 fragment ion, confirming the peptide sequence in high confidence as well as the site and mass of the glycosylation modification. An asterisk (*) represents carbamidomethylation of cysteine; at symbol (@) represents the site of N-glycosylation, respectively [55]
Fig. 7
Fig. 7
A MALDI-MS of released and permethylated O-glycans released from IgA. B Nanospray-full MS spectrum of released and permethylated O-glycans from human siat7e-modified MDCK cells. O-glycans were released by reductive β-elimination
Fig. 8
Fig. 8
Nanospray-FT full MS spectrum of permethylated N-glycans released from human serum. N-linked glycans were released enzymatically by PNGase F, permethylated and analyzed by a direct infusion into the mass spectrometer
Fig. 9
Fig. 9
Typical fragmentation pattern and nomenclature for A peptides and B glycans, during tandem mass spectrometry
Fig. 10
Fig. 10
Common representation of glycans. A CFG notation [46]. B Oxford-Dublin notation [47]
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