Recent advances in the MS analysis of glycoproteins: Theoretical considerations

Abstract

Protein glycosylation is involved in a broad range of biological processes that regulate protein function and control cell fate. As aberrant glycosylation has been found to be implicated in numerous diseases, the study and large-scale characterization of protein glycosylation is of great interest not only to the biological and biomedical research community, but also to the pharmaceutical and biotechnology industry. Due to the complex chemical structure and differing chemical properties of the protein/peptide and glycan moieties, the analysis and structural characterization of glycoproteins has been proven to be a difficult task. Large-scale endeavors have been further limited by the dynamic outcome of the glycosylation process itself, and, occasionally, by the low abundance of glycoproteins in biological samples. Recent advances in mass spectrometry (MS) instrumentation, and progress in miniaturized technologies for sample handling, enrichment and separation, have resulted in robust and compelling analysis strategies that effectively address the challenges of the glycoproteome. This review summarizes the key steps that are involved in the development of efficient glycoproteomic analysis methods, and the latest innovations that led to successful strategies for the characterization of glycoproteins and their corresponding glycans. As a follow-up to this work, we review innovative capillary and microfluidic-MS workflows for the identification, sequencing, and characterization of glycoconjugates.

Tandem Mass Spectrometry of Peptides

“…This PTM is important in protein function and cell fate differentiation. Glycosylation, like phosphorylation, is regulated by enzymes and it involves the binding of glycans to proteins being attached in linked or branched chains with various glycan composition and length (Lazar et al, 2011), hence having an impact on charge, conformation and stability of proteins. Glycosylation has been shown to have an important role in several diseases, including cancer, inflammatory diseases and congenital disorders, etc.…”

“…Glycosylation has been shown to have an important role in several diseases, including cancer, inflammatory diseases and congenital disorders, etc. The study of glycosylation of proteins has been useful for the development of novel vaccines and biomarkers discovery for diagnosis (Lazar et al, 2011). Table 2.…”

“…Just like with phosphorylation, glycosylation modified protein/peptides need to be enriched prior to mass spectrometry analysis. A common methodology for this purpose includes lectin enrichment, which can selectively separate complex carbohydrate structures, as reviewed by Lazar et al (2011). In mass spectrometry, the analysis of glycosylated peptides is particularly challenging, due to the heterogeneity of oligossacharide moieties and to proteolysis difficulties, since glycan motifs can prevent access of site-specific endoproteases, reducing protein coverage and identification.…”

“…The ionization of these modified peptides is also challenging due to the fact that they can have low ionization efficiency, which implies lower sensitivity in peptide detection. The ions generated can be due to the sequential loss of the sugar residues, however, the ions from the fragmentation of the peptide backbone can be in low abundance or even absent (Lazar et al, 2011). Glycation is a type of chemical modification that can occur in vivo, but it is not controlled enzymatically.…”

Immobilization of Styrene-acrylamide Co-polymer on Either Silica Particles or Inner Surface of Silica Capillary for the Separation of D-Glucose Anomers

“…Various glycosylation analysis strategies including pretreatments of samples, chromatography, and mass spectrometry have been well introduced in some review articles. [4][5][6][7][8][9][10][11] HPLC and HPCE methods have been developed for the high-resolution and reproducible quantitation of carbohydrates. 12,13 The high performance anion exchange chromatographypulsed amperometry (HPAEC-PAD) system was developed for common use in separation and detection of saccharides.…”