Background

Protein N-glycosylation – the attachment of glycans to asparagine residues – is a major posttranslational modification for regulating many key biological processes from membrane trafficking and protein degradation to immune response and cell-cell communication (Moremen et al., 2012). The importance of proper N-glycosylation in human health is underscored by the finding that mutations in the protein N-glycosylation machinery components cause congenital disorders of glycosylation with multi-system abnormalities (Freeze et al., 2015). Furthermore, increasing evidence links aberrant protein N-glycosylation to various human diseases including cancer, diabetes, and neurodegenerative diseases (Schedin-Weiss et al., 2014; Pinho and Reis, 2015; Reily et al., 2019).

A mechanistic understanding of the roles of protein N-glycosylation in biology and pathophysiology requires the system-wide elucidation of in vivo N-glycosylation sites (N-glycosites) on N-glycosylated proteins (N-glycoproteins) in health and disease. Protein N-glycosylation sites typically have a canonical sequon N-X-S|T, where X represents any amino acid except proline. However, whether a sequon-containing site can be N-glycosylated in vivo is dependent on protein folding and localization, site availability, and accessibility to N-glycosylation enzymes (Moremen et al., 2012; Cherepanova et al., 2016); thus, the in vivo N-glycosylation sites on glycoproteins have to be determined experimentally. Furthermore, since stress and pathophysiological conditions can alter N-glycosylation site occupancy and expose the cryptic N-glycosites (i.e., normally unutilized N-glycosylation sequons) for N-glycosylation (Pinho and Reis, 2015; Cherepanova et al., 2016), it is important to determine disease-associated, site-specific changes in protein N-glycosylation site occupancy to gain mechanistic insights into N-glycosylation dysregulation and its roles in disease pathogenesis.

High-resolution mass spectrometry-based glycoproteomics technologies provide powerful tools for unbiased, large-scale, site-specific N-glycoproteome profiling analyses of complex biological samples. For example, in our recently published study (Zhang et al., 2020), we used an N-glycoproteomics workflow consisting of SDS-mediated protein extraction and filter-aided sample preparation (FASP) (Wisniewski, 2017), zwitterionic chromatography-hydrophilic interaction chromatography (ZIC-HILIC)-based glycopeptide enrichment (Ma et al., 2015), ¹⁸O-labeling of in vivo N-glycosylation sites (Kuster and Mann, 1999), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to characterize protein N-glycosylation in human Alzheimer's disease (AD) and control brains. We identified 4730 ¹⁸O-labeled N-glycosite-containing peptides (hereafter referred to as N-glycopeptides) and mapped 2294 in vivo N-glycosylation sites on 1132 brain N-glycoproteins (Zhang et al., 2020).

With advanced N-glycoproteomics technologies enabling simultaneous, quantitative measurement of abundance profiles for thousands of N-glycopeptides and in vivo N-glycosylation sites, analysis of such large datasets has become a challenge. In our recent study (Zhang et al., 2020), we performed differential analysis of N-glycopeptide abundance and identified 118 N-glycopeptides with >1.3-fold change in N-glycopeptide abundance in AD as compared with control cases. By integrated analysis of our N-glycoproteome and proteome profiling data from the same brain samples (Zhang et al., 2018), we identified 77 N-glycosites on 60 N-glycoproteins with >1.3-fold changes in N-glycosylation site occupancy in AD versus control brains (Zhang et al., 2020). Furthermore, we performed qualitative assessment of whether an N-glycosite was exclusively occupied in either AD or control brains and identified 89 N-glycosites on 76 glycoproteins with a gain of N-glycosylation in AD and 12 N-glycosites on 11 glycoproteins with a complete loss of N-glycosylation in AD. In total, we identified 137 differentially N-glycosylated proteins in AD versus control brains, including 92 hyperglycosylated proteins containing N-glycosites with increased N-glycosylation site occupancy and/or a gain of N-glycosylation in AD, 39 hypoglycosylated proteins containing N-glycosites with decreased N-glycosylation site occupancy or a loss of N-glycosylation in AD, and 6 aberrantly glycosylated proteins containing both hyperglycosylated and hypoglycosylated N-glycosites. These analyses have identified disease signatures of altered N-glycopeptides, N-glycoproteins, and N-glycosylation site occupancy in AD and suggest new targets for AD biomarker development (Zhang et al., 2020).

Here, we provide a protocol that describes how to perform mass spectrometry-based, systems-level analysis of in vivo N-glycosylation sites on N-glycosylated proteins and their changes in human disease (Figure 1). The protocol also includes quantitation and differential analysis of N-glycopeptide abundance, in addition to integrative N-glycoproteome and proteome data analyses, to determine disease-associated changes in N-glycosylation site occupancy and identify differentially N-glycosylated proteins in human disease versus control samples. For more details on the use and execution of this protocol, please refer to our research article (Zhang et al., 2020). Similar strategies as described in this protocol should be broadly applicable to study proteome-wide N-glycosylation alterations in response to different cellular stresses or pathophysiological conditions in other organisms or model systems.

Figure 1. Overview of the experimental workflow. The experimental procedures for these steps are described in this protocol.