Glycopeptide analysis by mass spectrometry

Abstract

Glycosylation is one of the most important post-translational modifications found in nature. Identifying and characterizing glycans is an important step in correlating glycosylation structure to the glycan's function, both in normal glycoproteins and those that are modified in a disease state. Glycans on a protein can be characterized by a variety of methods. This review focuses on the mass spectral analysis of glycopeptides, after subjecting the glycoprotein to proteolysis. This analytical approach is useful in characterizing glycan heterogeneity and correlating glycan compositions to their attachment sites on the protein. The information obtained from this approach can serve as the foundation for understanding how glycan compositions affect protein function, in both normal and aberrant glycoproteins.

Comparison of HPLC/ESI-FTICR MS versus MALDI-TOF/TOF MS for glycopeptide analysis of a highly glycosylated HIV envelope glycoprotein

“…The glycosidic cleavage resulting from a loss of a HexNAc is represented by a square in present. The Y 1 ion is a useful characteristic product ion, [35] and it is observed in all MS/MS data of the glycopeptides subjected to ESI MS/MS experiments; since the Y 1 ion corresponds to the peptide attached to the innermost N-acetyl glucosamine (HexNAc), subtracting the mass of this sugar (203 Da) from the observed mass of the Y 1 ion provides the mass of the glycosylated peptide moiety. The product ion corresponding to the Y 1 ion can be identified either manually or by simply inputting the MS/MS peaklist for this glycopeptide (m/z 1477.15) into GlycoPep ID, available at http://hexose.chem.ku.edu/predictiontable2.php, which automatically outputs the potential peptide and its corresponding Y 1 ion (see the Experimental section).…”

A Comparison of Energy-Resolved Vibrational Activation/Dissociation Characteristics of Protonated and Sodiated High Mannose N-Glycopeptides

“…In particular, the analysis of protein glycosylation at the level of proteolytic glycopeptides is of high interest because, unlike methods which involve glycan release, this approach confers protein site specificity to analysis of the modifying glycans [5][6][7][8]. While advantageous in allowing for glycans of specific compositions and topologies to be localized to specific sites of a glycoprotein, these means of glycoprotein characterization can also be technically challenging since the heterogeneities and structural complexities of the oligosaccharide and the polypeptide must be addressed simultaneously [9][10][11].…”

Label-free quantitation: A new glycoproteomics approach

“…It is expected that other methods would be used to identify all the glycopeptides present, before using the quantitative method. Appropriate complementary techniques for identifying glycopeptide compositions are welldescribed in the literature [12,17,20,21,40,43,45,46]. The second major limitation of this method is that it is only applicable to glycoproteins containing neutral glycoforms; a mixture of sialylated glycoforms, or other negatively charged species, and neutral glycans typically requires the use of both positive and negative mode to accurately identify the glycoforms present [40].…”

“…While glycan analysis is clearly a more established technique [15][16][17][18], this approach restricts the amount of information one can obtain about the glycosylation profile. For example, in purified, multiply glycosylated proteins, the study of released glycans would only provide aggregate information about the glycosylation on a protein, and it would not provide information about the glycosylation profile at a specific glycosylation site.…”

“…This introduces many problems in biomarker discovery, such as not being able to identify whether or not the glycan's concentration increased because a protein containing that glycan was overexpressed, or if one or several proteins' glycosylation profile changed, causing the glycan to be more abundant, even though the protein level(s) are not altered [4]. In contrast to glycan analysis, glycopeptide analysis provides glycosylation site-specific information for purified proteins [17,20,21], and it could potentially be useful in distinguishing between glycosylation changes and protein expression changes, since the protein information is encoded in the glycopeptide. For the reasons described herein, we are pursuing quantitative methods for glycopeptides.…”