Combining Results from Lectin Affinity Chromatography and Glycocapture Approaches Substantially Improves the Coverage of the Glycoproteome

Abstract

Identification of glycosylated proteins, especially those in the plasma membrane, has the potential of defining diagnostic biomarkers and therapeutic targets as well as increasing our understanding of changes occurring in the glycoproteome during normal differentiation and disease processes. Although many cellular proteins are glycosylated they are rarely identified by mass spectrometric analysis (e.g. shotgun proteomics) of total cell lysates. Therefore, methods that specifically target glycoproteins are necessary to facilitate their isolation from total cell lysates prior to their identification by mass spectrometrybased analysis. To enrich for plasma membrane glycoproteins the methods must selectively target characteristics associated with proteins within this compartment. We demonstrate that the application of two methods, one that uses periodate to label glycoproteins of intact cells and a hydrazide resin to capture the labeled glycoproteins and another that targets glycoproteins with sialic acid residues using lectin affinity chromatography, in conjunction with liquid chromatography-tandem mass spectrometry is effective for plasma membrane glycoprotein identification. We demonstrate that this combination of methods dramatically increases coverage of the plasma membrane proteome (more than one-half of the membrane glycoproteins were identified by the two methods uniquely) and also results in the identification of a large number of secreted glycoproteins. Our approach avoids the need for subcellular fractionation and utilizes a simple detergent lysis step that effectively solubilizes membrane glycopro-

In-depth research of multidrug resistance related cell surface glycoproteome in gastric cancer

“…Secondly, the membrane complex was not completely destroyed during cell lysis and glycoprotein enrichment. Thirdly, high abundance of some non-specific proteins from cell lysate binding to hydrazide resin was proven to occur even in stringent washing conditions in McDonald's experiment [39]. Additionally, not only low abundance of glycoprotein, but also many low abundance of non-specific bound proteins can be identified by using 2D LC-MS/MS.…”

Cocapture of cognate and bystander antigens can activate autoreactive B cells

“…One possibility, for example, is that the interaction we observe here depends on the interaction between sialic acid residues on the MOG protein and the sialic acid-binding domain of the HA (17). This would limit the phenomenon to the set of sialated proteins, which includes much but not all of the membrane proteome (18). A major function of viral glycoproteins is attachment to host cell membranes, and therefore, the likelihood that viral proteins bind to the extracellular domains of membrane proteins is high.…”

Quantitative N-linked Glycoproteomics of Myocardial Ischemia and Reperfusion Injury Reveals Early Remodeling in the Extracellular Environment

“…Large-scale glycoproteomic profiling has proven challenging, and only a handful of methods have been developed to investigate specific glycan-containing glycopeptides, for example lectin affinity for specific sugar subunits (12) or titanium dioxide (TiO 2 ) for sialic acids (SA) 1 (13). A comprehensive glycoproteomic profile most likely requires multiple fractionation, enrichment and mass spectrometric procedures applied in parallel.…”