Comprehensive analysis of protein glycosylation by solid-phase extraction of N-linked glycans and glycosite-containing peptides

Abstract

Comprehensive characterization of protein glycosylation is critical for understanding the structure and function of glycoproteins. However, due to the complexity and heterogeneity of glycoprotein conformations, current glycoprotein analyses focus mainly on either the de-glycosylated glycosylation site (glycosite)-containing peptides or the released glycans. Here, we describe a chemoenzymatic method called solid phase extraction of N-linked glycans and glycosite-containing peptides (NGAG) for the comprehensive characterization of glycoproteins that is able to determine glycan heterogeneity for individual glycosites in addition to providing information about the total N-linked glycan, glycosite-containing peptide and glycoprotein content of complex samples. The NGAG method can also be applied to quantitatively detect glycoprotein alterations in total and site-specific glycan occupancies.

Simultaneous quantification of N- and O-glycans using a solid-phase method

“…In our laboratory, GIG has been used for studies of N-glycan profiling in tissue or blood samples derived from patients with prostate cancer 44 , pancreatic cancer 25 , ovarian cancer 45 and cardiac hypertrophy 46 , as well as samples from glycoengineered Chinese hamster ovary (CHO) cells 47 , and for studies of glycoforms of HIV gp 120 (ref. 48), glycoengineered sialylation of CHO cells 49 and N-gly-cosylation in cockroach allergen regulation of human basophil function 50 .…”

Classification of Tandem Mass Spectra for Identification of N- and O-linked Glycopeptides

“…Previously, these studies mainly involved enrichment or isolation of glycopeptides using hydrazide chemistry, lectin affinity techniques or hydrophilic interaction chromatography (HILIC), which result in identification of glycans and glycosite-containing peptides1112131415. However, site-specific glycosylation microheterogeneity plays key roles in the function of this post-translational modification16171819 and therefore the ability to preserve this information is critical. Hitherto advances in mass spectrometry techniques has facilitated the detection of intact glycopeptides from recombinant proteins, glycoprotein cocktails or complex biological samples1820212223242526.…”

“…However, site-specific glycosylation microheterogeneity plays key roles in the function of this post-translational modification16171819 and therefore the ability to preserve this information is critical. Hitherto advances in mass spectrometry techniques has facilitated the detection of intact glycopeptides from recombinant proteins, glycoprotein cocktails or complex biological samples1820212223242526. However, mass spectrometry data is being generated at a rate as high as one gigabytes/hour on a single instrument, resulting in massive amounts of glycoproteomics data being collected.…”

“…Many efforts in the glycoproteomics field have focused on N-glycosylation18202324252627. Higher-energy collisional dissociation (HCD) fragments the glycopeptides both at glycosidic and peptide bonds, generating glycan oxonium ions, peptide fragment ions and peptide ions with partial glycans26.…”

“…Higher-energy collisional dissociation (HCD) fragments the glycopeptides both at glycosidic and peptide bonds, generating glycan oxonium ions, peptide fragment ions and peptide ions with partial glycans26. This fragmentation method, alone or in combination with others, has proven to be effective in fragmentation and identification of N-glycoproteins1826282930. However, the use of HCD for analysis of O-glycoproteins remains largely unexplored.…”