Hong, Q., et al. (2015). “A Method for Comprehensive Glycosite-Mapping and Direct Quantitation of Serum Glycoproteins.” J Proteome Res.
A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and non-specific enzyme digestions followed by nano LC-Chip/QTOF mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20-min UPLC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantitation method that involves protein enrichments. The absolute protein quantitation has a wide linear range (3-4 orders of magnitude) and low limit of quantitation (femtomole level). This rapid and robust quantitation technique, which provides quantitative information for both proteins and glycosylation, will further facilitate disease biomarker discoveries.
Krishnan, S., et al. (2015). “Combined HDL proteomic and glycomic profiles in patients at risk for coronary artery disease..” J Proteome Res.
OBJECTIVES: To test whether recently developed methods for comprehensive profiling of the HDL glycome combined with the HDL proteome can distinguish individuals with coronary artery disease (CAD) from those without. METHODS: Twenty subjects, at risk for CAD, who underwent diagnostic coronary arteriography were analyzed. Ten subjects had CAD, and ten did not. HDL were extracted from fasting plasma samples by ultracentrifugation, followed by shotgun proteomic, glycomic and ganglioside analyses using LC-MS. CAD vs. non-CAD subjects’ data were compared using univariate and multivariate statistics. RESULTS: Principal components analysis showed a clear separation of CAD and non-CAD subjects, confirming that combined HDL proteomic and glycomic profiles distinguished at-risk subjects with atherosclerosis from those without. CAD patients had lower HDL apolipoprotein content (specifically Apo AI, AII and E, p <0.05), and lower serum amyloid A2 (SAA2, p = 0.020) and SAA4 (p = 0.007) but higher sialylated glycans (p<0.05). CONCLUSION: Combined proteomic and glycomic profiling of isolated HDL was tested as a novel analytical approach for developing biomarkers of disease. In this pilot study we found that HDL proteome and glycome distinguished between individuals who had CAD from those who did not within a group of individuals equally at risk for heart disease.
Park, D., et al. (2015). “Characteristic Changes in Cell Surface Glycosylation Accompany Intestinal Epithelial Cell (IEC) Differentiation: High Mannose Structures Dominate the Cell Surface Glycome of Undifferentiated Enterocytes.” Mol Cell Proteomics.
Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Due to their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract.