Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins

Abstract

We describe here a strategy for the large-scale identification of N-glycosylated proteins from a complex biological sample. The approach, termed isotope-coded glycosylation-site-specific tagging (IGOT), is based on the lectin column-mediated affinity capture of a set of glycopeptides generated by tryptic digestion of protein mixtures, followed by peptide-N-glycosidase-mediated incorporation of a stable isotope tag, 18O, specifically into the N-glycosylation site. The 18O-tagged peptides are then identified by multi-dimensional liquid chromatography-mass spectrometry (LC-MS)-based technology. The application of this method to the characterization of N-linked high-mannose and/or hybrid-type glycoproteins from an extract of Caenorhabditis elegans proteins allowed the identification of 250 glycoproteins, including 83 putative transmembrane proteins, with the simultaneous determination of 400 unique N-glycosylation sites. Because the method is applicable to the systematic identification of a wide range of glycoproteins, it should facilitate basic glycobiology research and may be useful for diagnostic applications, such as genome-wide screening for disease-related glycoproteins.

Solid-phase extraction of N-linked glycopeptides

“…However, studies on protein glycosylation have been complicated by the diverse structure of protein glycans and the lack of effective tools to identify the protein glycosylation site and glycan structure. For example, human protein database contains thousands of predicted N-linked glycosylated proteins, however, before high-throughput methods for the identification of glycosites were developed in 2003, only 172 proteins were proven to be glycoproteins experimentally in the Protein Information Resources-Protein Sequence Database (http://pir.georgetown.edu/pirwww/search/textpsd.shtml) 4 .…”

“…Recently, two new methods for isolation and identification of N-linked glycopeptides in complex biological samples--solidphase extraction of N-linked glycopeptides (SPEG) and glycopeptide capture using lectin-affinity column chromatography--have been reported 4,5 . In both instances, mass spectrometry is used after the isolation of the N-glycopeptides to identify the N-glycosites and quantify the relative abundance of glycopeptides using isotope-coded tags.…”

“…After elution of glycopeptides from lectin column, peptide-N-glycosidase is then used to release the N-glycopeptides and the resulting peptides are subjected to mass spectrometry to identify the N-linked glycopeptides and sites of glycosylation 4 . For glycopeptide capture using hydrazide chemistry, peptides containing either N-linked or O-linked oligosaccharides are conjugated to a solid support covalently.…”

A method to determine the ionization efficiency change of peptides caused by phosphorylation

“…In this respect, an isotope-labeled internal standard was used to achieve absolute quantification of PTMs in proteins. For instance, isotope-coded affinity tag (ICAT) [3], stable isotope labeling with amino acids in cell culture (SILAC) [4], and introducing 18 O labeling by enzymatic digestion in H 2 18 O [5] have been employed. In addition, an isotope-dilution method, where the absolute or relative quantification of PTM levels of peptides is achieved by doping a known amount of the synthetic isotope-labeled peptides into the peptide samples as the internal standard, was reported [6].…”

Large‐scale N‐glycoproteome map of rat brain tissue: Simultaneous characterization of insoluble and soluble protein fractions

“…Therefore, high efficient and specific enrichment procedure is imperative for large-scale glycoproteome analysis. Recently, various methods, including hydrophilic interaction liquid chromatography [5,6], lectinbased affinity chromatography [7][8][9], hydrazide chemistry [10] and TiO 2 [11,12], have been developed, and further used for the selective enrichment of glycoproteins/glycopeptides. Among above-mentioned methods, Hydrazide gel-based solid-phase extraction becomes a popular technique, mainly due to its high efficiency and excellent selectivity, and has been used for the analysis of N-glycoproteome in various biological samples [10,[13][14][15].…”