Glycoproteins Enrichment and LC-MS/MS Glycoproteomics in Central Nervous System Applications

Abstract

Proteins and glycoproteins play important biological roles in central nervous systems (CNS). Qualitative and quantitative evaluation of proteins and glycoproteins expression in CNS is critical to reveal the inherent biomolecular mechanism of CNS diseases. This chapter describes proteomic and glycoproteomic approaches based on liquid chromatography/tandem mass spectrometry (LC-MS or LC-MS/MS) for the qualitative and quantitative assessment of proteins and glycoproteins expressed in CNS. Proteins and glycoproteins, extracted by a mass spectrometry friendly surfactant from CNS samples, were subjected to enzymatic (tryptic) digestion and three down-stream analyses: (1) a nano LC system coupled with a high resolution MS instrument to achieve qualitative proteomic profile, (2) a nano LC system combined with a triple quadrupole MS to quantify identified proteins, and (3) glycoprotein enrichment prior to LC-MS/MS analysis. Enrichment techniques can be applied to improve coverage of low abundant glycopeptides/glycoproteins. An example described in this chapter is hydrophilic interaction liquid chromatographic (HILIC) enrichment to capture glycopeptides, allowing efficient removal of peptides. The combination of three LC-MS/MS-based approaches is capable of the investigation of large-scale proteins and glycoproteins from CNS with an in-depth coverage, thus offering a full view of proteins and glycoproteins changes in CNS.

The glycosylation in SARS-CoV-2 and its receptor ACE2

“… 120 – 123 Glycans usually need to be enriched by hydrophilic materials, such as porous graphitized carbon (PGC), 124 , 125 before MS analysis, 126 – 128 while intact glycopeptides can be analyzed by MS with or without enrichment, 129 although the enrichment step is beneficial for deep characterization of glycopeptides with low stoichiometry. 130 – 145 Historically, chemical enrichment methods were adopted for both glycans and glycopeptides such as hydrazide chemistry, 146 – 149 boronic acid, 142 , 150 – 152 etc. Hydrophilic interaction liquid chromatography (HILIC), 100 , 153 – 155 lectin affinity chromatography, 121 , 156 , 157 and graphitized carbon chromatography are the most widely adopted methods for enrichment of glycopeptides.…”

Protein Glycosylation Investigated by Mass Spectrometry: An Overview

“…HILIC separation takes advantage of polar interactions between the hydroxy groups of glycans and the stationary phase. The efficient removal of the non-glycosylated counterpart takes place by using organic solvent washing followed by glycopeptide elution with an aqueous buffer as detailed in a study on the central nervous system glycoproteomics [103]. This method allows the glycopeptides separations based on their oligosaccharide portion, and its results are extremely useful when there is more than one glycosylation on the same peptide moiety.…”

Profiling of 54 plasma glycoproteins by label-free targeted LC-MS/MS

“…Given their high sensitivity and selectivity, mass spectrometry-based methods are widely used to identify and quantify glycoproteins, recognise glycosylation sites, and characterise the attached oligosaccharides [16]. Targeted glycoprotein quantification by LC-MS/MS provides an improved sensitivity and specificity when compared to shotgun glycoproteomics workflows [7,17]. A targeted approach can be used for relative or absolute protein quantification applying either stable isotope standards (SIS), reference-labelled peptides or label-free workflows [18].…”