Glycoprotein Enrichment Analytical Techniques: Advantages and Disadvantages

Abstract

Protein glycosylation is one of the most important posttranslational modifications. Numerous biological functions are related to protein glycosylation. However, analytical challenges remain in the glycoprotein analysis. To overcome the challenges associated with glycoprotein analysis, many analytical techniques were developed in recent years. Enrichment methods were used to improve the sensitivity of detection, while HPLC and mass spectrometry methods were developed to facilitate the separation of glycopeptides/proteins and enhance detection, respectively. Fragmentation techniques applied in modern mass spectrometers allow the structural interpretation of glycopeptides/proteins, while automated software tools started replacing manual processing to improve the reliability and throughput of the analysis. In this chapter, the current methodologies of glycoprotein analysis were discussed. Multiple analytical techniques are compared, and advantages and disadvantages of each technique are highlighted.

Keywords: Enrichment; Glycoprotein; Glycoproteomics; LC-MS/MS; Proteomics.

Figure 7.1. Flowchart highlighting typical glycoproteomics work flow

Glycopeptide enrichment techniques: A. lectin affinity, B. hydrazide chemistry, C. immunoaffinity, D. HILIC, and E. click-chemistry. Figure reproduced with permission (Song et al., 2015).

Figure 7.2. Boronic acid enrichment and electrostatic repulsion hydrophilic interaction chromatography enrichment

A. A covalent bond between the stationary media and the glycan hydroxyl groups allows for clean separations. Reproduced with permission (Ongay et al., 2012). B. Silica beads functionalized with polyethyleneimine attract uncharged glycans (right) while repelling charged glycans (left). Reproduced with permission (L. H. Zacharias, A. ; Song, E.; Zhao, J.; Zhu, R.; Mirzaei, P.; Mechref, Y., 2016).

Figure 7.3. Comparison of different glycopeptide enrichment methods

A. The Venn diagram depicts the number of identified unique glycosylation sites for HILIC and HILIC-ERLIC enrichment methods (shown bolded in the parentheses is the total number of glycopeptides identified in each case) and of those, which glycosylation sites were unique to the individual methods. Reproduced with permission (L. G. Zacharias et al., 2016). B. Comparisons of MRM quantitation for 17 common glycosylation site between lectin affinity and hydrazide chemistry enrichments in terms of ratio between different groups of biological samples. (DF: disease-free, HGD: high grade dysplasia, EAC: esophageal adenocarcinoma.) Reproduced with permission (Song et al., 2014).

Figure 7.4. Separation of glycopeptides derived from porcine thyroglobulin (PTG) with C18 analytical column

The same peptide backbone is associated with multiple glycan residues highlighting the microheterogeneity at this glycosylation site. Reproduced with permission (J. Zhao et al., 2016).

Figure 7.5. Separation and analysis of glycopeptides by Ion Mobility Mass Spectrometry

A. Traveling Wave Ion Mobility Mass Spectrometry (TWIMS) identified multiple conformers of the isobaric Muc2 glycopeptides (PTTTPITTTTTVTPTPTPTGTQT with GalNAc 19 and GlcNac 20). TWIMS was not able to differentiate between the two intact glycopeptides; B. however, after CID, TWIMS-MS could distinguish the diagnostic oxonium ions from each Mucin glycopeptide. C. High-field asymmetric wave ion mobility mass spectrometry (FAIMS-MS) separation of two isobaric O-linked glycopeptides, differing only in glycan site attachment. D, E. glycopeptide identity confirmed with ETD MS²; diagnostic c and z ions are indicated in blue and red.

Figure 7.6. Capillary electrophoresis mass spectrometry (CE-MS) of therapeutic antibody

CE-MS quantified 14-glycoforms on one glycopeptide from Trastuzumab. Reproduced with permission (Lew et al., 2015).

Figure 7.7. Combined top-down and middle-down proteomics approach to identifying IgG glycosylation profile

A Top-down native mass spectrometry of intact IgG to readily identify glycoforms. B Middle-down mass spectrometry to identify peptide backbone and any post-translational modifications. Reproduced with permission (Tran et al., 2016).