Mapping human N-linked glycoproteins and glycosylation sites using mass spectrometry

Abstract

N-linked glycoprotein is a highly interesting class of proteins for clinical and biological research. Over the last decade, large-scale profiling of N-linked glycoproteins and glycosylation sites from biological and clinical samples has been achieved through mass spectrometry-based glycoproteomic approaches. In this paper, we reviewed the human glycoproteomic profiles that have been reported in more than 80 individual studies, and mainly focused on the N-glycoproteins and glycosylation sites identified through their deglycosylated forms of glycosite-containing peptides. According to our analyses, more than 30,000 glycosite-containing peptides and 7,000 human glycoproteins have been identified from five different body fluids, twelve human tissues (or related cell lines), and four special cell types. As the glycoproteomic data is still missing for many organs and tissues, a systematical glycoproteomic analysis of various human tissues and body fluids using a uniform platform is still needed for an integrated map of human N-glycoproteomes.

Atypical N-glycosylation of SARS-CoV-2 impairs the efficient binding of Spike-RBM to the human-host receptor hACE2

“…In consequence, validated evidences of atypical N-glycan occupancy have been reported for major human serum glycoproteins such as von Willebrand Factor, CD69, serotransferrin, factor XI, albumin, and α−1B-glycoprotein 15,26,27 . Furthermore, these atypical N-glycosylations have a widespread presence in the proteome of human individuals 15,26,27 . Nonetheless, in the context of the COVID-19 pandemic, it remains elusive whether these atypical N-glycosylation sequons are also encoded in the genome of the original Wuhan-Hu-1 (the novel coronavirus), and how they could potentially contribute to the virus tropism of the SARS-CoV-2 variants of major concern.…”

“…By using the SnapGene® software (from GSL Biotech; available at snapgene.com), we manually identified and curated all protein-encoding genes and other relevant traits on SARS-CoV-2 reference genome, according to the updated COVID-19 scientific information publicly available in the literature. Similarly, we performed a genome-wide atypical N-glycosylation site identification in all the SARS-CoV-2 protein-encoding sequences, especially in SGP, according to the sequon motifs NXC and NXV 14,15,26,27 , including the nonfunctional motifs NPC and NPV (Table 1 and Figure 1). For comparative analysis, we also confirmed the reported prediction of the 22 canonical N-linked glycosylation sites in the SARS-CoV-2 spike-glycoprotein sequence 12,24 , by using the online web-server (http://www.cbs.dtu.dk/services/NetNGlyc), choosing a predication threshold of 0.5.…”

“…The copyright holder for this preprint (which this version posted April 10, 2021. ; https://doi.org/10.1101/2021.04.09.439154 doi: bioRxiv preprint and NXV motifs 14,15,26,27 . In consequence, validated evidences of atypical N-glycan occupancy have been reported for major human serum glycoproteins such as von Willebrand Factor, CD69, serotransferrin, factor XI, albumin, and α−1B-glycoprotein 15,26,27 . Furthermore, these atypical N-glycosylations have a widespread presence in the proteome of human individuals 15,26,27 .…”

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

“…MS-based Glycoproteomic Enrichment Strategies 8 analysis. We direct readers to prior reviews on glycoproteomic enrichment strategies for more historical perspectives (42-50), and we recommend several reviews that focus on N-or Oglycosylation classes more specifically (51)(52)(53)(54)(55)(56)(57)(58)(59). We also recognize recent reviews that more categorically address glycopeptide fragmentation methods (60)(61)(62), quantitative strategies for glycoproteomics (63,64), and informatics tools (65-69) -all of which make glycoproteomics an exciting and burgeoning field (70)(71)(72)(73).…”

Site‐ and structure‐specific characterization of the human urinary N‐glycoproteome with site‐determining and structure‐diagnostic product ions

“…Aberrant N‐glycosylation has been widely linked to malignant diseases like cancers 1–9 . With advancements in enrichment, chromatographic separation, tandem mass spectrometry (MS/MS), and bioinformatics, MS‐based N‐glycoproteomics has been widely adopted for characterizing the N‐glycoproteome at the intact N‐glycopeptide level 10–35 . Our recent development of the intact N‐glycopeptide search engine GPSeeker has made it possible to perform large‐scale structural N‐glycoproteomics characterization with structure‐diagnostic product ions to distinguish N‐glycan isomers with the same monosaccharide compositions but different sequences/linkages 36,37 …”