An enrichment method based on synergistic and reversible covalent interactions for large-scale analysis of glycoproteins

Xiao, Haopeng; Chen, Weixuan; Smeekens, Johanna M.; Wu, Ronghu

doi:10.1038/s41467-018-04081-3

Cited by 133 publications

(126 citation statements)

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“…We compared the glycopeptide and non-glycopeptide fractions in each parallel replicate, and showed that the specificity of our method is 55.4%. Our performance was better than the specificity of previously boronic acid and ZIC-HILIC enrichment method 8 . After N-glycan was released by the endoglycosidase, the core fucose was retained on the core GlcNAc residue, which allowed us to simultaneously distinguish the non-fucosylated and core fucosylated peptides.…”

Section: Resultsmentioning

confidence: 54%

“…However, due to the low abundance of glycosylated peptides and the heterogeneity of glycan structures, N-glycopeptide enrichment is required. Several enrichment methods have been reported, including lectin 5 and hydrazide chemistry-based methods 6,7 , boronic acid enrichment 8 , hydrophilic interaction liquid chromatography (HILIC) 9 and metabolic labeling 10,11 . In general, these strategies for detecting N-linked glycosylated sites require an additional de-glycosylation step by N-glycosidase F (PNGase F) before MS detection 5–7 .…”

Section: Introductionmentioning

confidence: 99%

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A Chemoenzymatic Method for Glycoproteomic N-glycan Type Quantitation

Cheng

et al. 2019

Preprint

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29Glycosylation is one of the most important post-translational modifications in biological systems. 30Current glycoproteome methods mainly focus on qualitative identification of glycosylation sites or intact 31 glycopeptides. However, the systematic quantitation of glycoproteins has remained largely unexplored. 32Here, we developed a chemoenzymatic method to quantitatively investigate N-glycoproteome based on 33 the N-glycan types. Taking advantage of the specificity of different endoglycosidases and isotope 34 dimethyl labeling, six N-glycan types of structures linked on each glycopeptide, including high-35 mannose/hybrid, bi-antennary and tri-antennary with/without core fucose, were quantified. As a proof of 36 principle, the glycoproteomic N-glycan type quantitative (glyco-TQ) method was first used to determine 37 the N-glycan type composition of immunoglobulin G1 (IgG1) Fc fragment. Then we applied the method 38 to analyze the glycan type profile of proteins in the breast cancer cell line MCF7, and quantitatively 39 revealed the N-glycan type micro-heterogeneity at both the glycopeptide and glycoprotein levels. The 40 novel quantitative strategy to evaluate the relative intensity of the six states of N-glycan type 41 glycosylation on each site provides a new avenue to investigate function of glycoproteins in broad areas, 42 such as cancer biomarker research, pharmaceuticals characterization and anti-glycan vaccine 43 development. 44 45 46 47 3 48Glycosylation is one of the most common post-translational modifications (PTM) 1 . Glycans exhibit 49 vast structural microheterogeneity which is mainly generated by variable glycan structures at each of 50 their specific glycosylation sites. The N-linked glycans are generally attached to the Asn at Asn-X- 51Ser/Thr consensus sequence, where X is any amino acid other than Pro 2 . The biosynthesis of N-linked 52 glycoproteins is under a complex sequence of enzymatically catalyzed events, leading to a variety of 53 diverse N-glycan structures. The diverse N-glycan structures are generally classified into three types: 54 high mannose, hybrid, and complex type glycans, with all N-glycans sharing a common penta-saccharide 55 (GlcNAc2 Man3) core structure 3 . Although the structure of glycan is variable, evidence shows that the 56 mammalian glycans are remarkably well conserved in certain organisms, expressing a distinct array of 57 glycan profiles under defined conditions 4 . 58Mass spectrometry (MS) is a powerful platform to comprehensively analyze protein glycosylation. 59However, due to the low abundance of glycosylated peptides and the heterogeneity of glycan structures, 60 N-glycopeptide enrichment is required. Several enrichment methods have been reported, including 61 lectin 5 and hydrazide chemistry-based methods 6,7 , boronic acid enrichment 8 , hydrophilic interaction 62 liquid chromatography (HILIC) 9 and metabolic labeling 10,11 . In general, these strategies for detecting N-63 linked glycosylated sites require an additional de-glycosylation step by N-glycosid...

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Section: Resultsmentioning

confidence: 54%

Section: Introductionmentioning

confidence: 99%

A Chemoenzymatic Method for Glycoproteomic N-glycan Type Quantitation

Cheng

et al. 2019

Preprint

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“…They later devised a boronic acid-based enrichment strategy to universally analyze glycoproteins, and identified 816 N-glycosylation sites from 332 glycoproteins in yeast. In a recent report, with more effective enrichment of glycopeptides, over 1,000 protein N-glycosylation sites were identified on over 500 proteins (Xiao et al, 2018c). Xiao et al (2016b) quantified the proteome and glycoproteome changes in yeast cells treated with tunicamycin compared with untreated cells.…”

Section: A Yeastmentioning

confidence: 99%

“…Strong interactions between boronic acid and glycans are critical to catch glycopeptides/proteins with low abundance. In order to enhance the interactions between boronic acid and glycans, different boronic acid derivatives were examined (Xiao et al, ). Among the derivatives tested, benzoboroxole was the most effective one, and it allowed us to identify the largest number of unique N‐glycopeptides from human whole cell lysates, as shown in Figure .…”

Section: Global Analysis Of Glycoproteinsmentioning

confidence: 99%

Global and site‐specific analysis of protein glycosylation in complex biological systems with Mass Spectrometry

Xiao

Sun

Suttapitugsakul

et al. 2019

Mass Spectrometry Reviews

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Protein glycosylation is ubiquitous in biological systems and plays essential roles in many cellular events. Global and site‐specific analysis of glycoproteins in complex biological samples can advance our understanding of glycoprotein functions and cellular activities. However, it is extraordinarily challenging because of the low abundance of many glycoproteins and the heterogeneity of glycan structures. The emergence of mass spectrometry (MS)‐based proteomics has provided us an excellent opportunity to comprehensively study proteins and their modifications, including glycosylation. In this review, we first summarize major methods for glycopeptide/glycoprotein enrichment, followed by the chemical and enzymatic methods to generate a mass tag for glycosylation site identification. We next discuss the systematic and quantitative analysis of glycoprotein dynamics. Reversible protein glycosylation is dynamic, and systematic study of glycoprotein dynamics helps us gain insight into glycoprotein functions. The last part of this review focuses on the applications of MS‐based proteomics to study glycoproteins in different biological systems, including yeasts, plants, mice, human cells, and clinical samples. Intact glycopeptide analysis is also included in this section. Because of the importance of glycoproteins in complex biological systems, the field of glycoproteomics will continue to grow in the next decade. Innovative and effective MS‐based methods will exponentially advance glycoscience, and enable us to identify glycoproteins as effective biomarkers for disease detection and drug targets for disease treatment. © 2019 Wiley Periodicals, Inc. Mass Spec Rev 9999: XX–XX, 2019.

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“…7,8 However, most of the existing methods rely on antibodies and uorescence probes to investigate individual proteins, which limit their capacities for large-scale analysis of protein degradation. In recent years, mass spectrometry (MS)-based proteomics has provided an opportunity to comprehensively characterize proteins, [9][10][11][12][13][14][15][16][17][18][19][20][21] including protein dynamics. [22][23][24][25] For instance, Doherty et al studied the stability of nearly 600 proteins in human A549 cells by using a dynamic stable isotope labeling by amino acids in cell culture (SILAC) approach.…”

Section: Introductionmentioning

confidence: 99%