Recent advances in mass spectrometric analysis of glycoproteins

Abstract

Glycosylation is one of the most common posttranslational modifications of proteins that plays essential roles in various biological processes, including protein folding, host-pathogen interaction, immune response, and inflammation and aberrant protein glycosylation is a well-known event in various disease states including cancer. As a result, it is critical to develop rapid and sensitive methods for the analysis of abnormal glycoproteins associated with diseases. Mass spectrometry (MS) in conjunction with different separation methods, such as capillary electrophoresis (CE), ion mobility (IM), and high performance liquid chromatography (HPLC) has become a popular tool for glycoprotein analysis, providing highly informative fragments for structural identification of glycoproteins. This review provides an overview of the developments and accomplishments in the field of glycomics and glycoproteomics reported between 2014 and 2016.

Keywords: Biomedical applications; Enrichment; Glycomics; Glycoproteomics; Mass spectrometry; Purification.

Figure 1

NaClO treatment of glycoproteins to release N-glycans. (a) Chemical scheme of NaClO treatment. (b) MALDI-TOF-MS profiles of N-glycans released from several glycoproteins by bleach treatment. (c) MALDI-TOF-MS profiles of permethylated glycans released from fetuin by PNGase F digestion (top) or bleach treatment (bottom). Reprint with permission from Ref [17]

Figure 2

(I) Workflow scheme of the purification of haptoglobin from serum using 96-well plate format for MS analysis of N-glycans. (II) MALDI-QIT-TOF MS spectra show the difference of fucosylation in tri- and tetra-antennary N-glycans of haptoglobin between (a) HCC and (b) cirrhosis. Reprint with premssion from Ref [24]

Figure 2

(I) Workflow scheme of the purification of haptoglobin from serum using 96-well plate format for MS analysis of N-glycans. (II) MALDI-QIT-TOF MS spectra show the difference of fucosylation in tri- and tetra-antennary N-glycans of haptoglobin between (a) HCC and (b) cirrhosis. Reprint with premssion from Ref [24]

Figure 3

(I) Selective enrichment of glycans using carbon-functionalized ordered graphene/mesoporous silica to further characterization by MS analysis. (II) MALDI-TOF MS analysis of N-linked glycan released from OVA: (a) without enrichment; (b) after enrichment by active carbon materials; (c) after enrichment by C-graphene@mSiO₂ nanocomposites. Reprint with permission from Ref [30]

Figure 4

Reaction mechanism of PNGase F and N-linked glycopeptides.

Figure 5

(I) HILIC-FLR-MS of (a) RFMS- and (b) IAB-labeled N-glycans from anticitrinin murine IgG1 (using 0.4 μg of glycoprotein). Fluorescence (FLR) chromatograms are shown in orange and base peak intensity (BPI) MS chromatograms are shown in blue. (c) Response factors for RFMS and IAB labeled glycan (measured as the FA2 peak area per sample of N-glycans resulting from 1 μg of anticitrinin murine IgG1) (II) Relative (%) performance of glycan labels. Response factors shown as percentages versus the fluorescence and MS response factors of RFMS labeled N-glycans. Reprint with permission from Ref [18]

Figure 6

(I) Summary of aminoxyTMT sample preparation steps. (II) (a) Extracted ion chromatograms of precursor ions for all six glycan structures in fetuin from bovine serum. (b) MS spectrum for the biantennary disialylated glycan. (c) MS² spectrum of the biantennary disialylated glycan. Reprint with permission from Ref [45]

Figure 7

(I) Illustration of mass spectrometry imaging of N-linked glycans from FFPE mouse brain tissue. (II) MALDI-MS analysis of N-glycans released by PNGase F from FFPE tissue section. (a) PNGase F negative and (b) PNGase F positive. Reprint with permission from Ref [50]

Figure 8

IM-MS separation of the isobaric glycopeptides GP1 and GP2, which can be distinguished based on their drift time (top and middle) and separated in mixtures (bottom). Reprint with permission from Ref [67]

Figure 9

(a) Quantitation of intact recombinant antibody product variants using standard addition approach by mass spectrometry. (b) Representative figures for MS data extraction, deconvolution, and normalization. Reprint with permission from Ref [75]

Figure 10

Examples of identified glycoforms at N102 glycosylation site using CID MS/MS and ETD MS/MS. The assignment of fragments of HexNAc2Hex5 on HSLFHPEDTGQVFQVSHSFPHPLYN102MSLLK with m/z value of 942.6390 in CID MS/MS (A) and ETD MS/MS (B). The assignment of fragments of HexNAc2Hex5 on HSLFHPEDTGQVFQVSHSFPHPLYN102MCAMSLLK with m/z value of 795.2017 in CID MS/MS (C) and in ETD MS/MS (D). The fragments indicating N102 glycosylation and the carbamidomethylation of methionine are highlighted in yellow. Reprint with permission from Ref [95]