Stable isotopic labeling-based quantitative targeted glycomics (i-QTaG).

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling-based quantitative targeted glycomics (i-QTaG) technique for the comparative and quantitative analysis of total N-glycans using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N-glycans using a model glycoprotein (bovine fetuin). Moreover, the i-QTaG using MALDI-TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of (13) C6 /(12) C6 -2-aminobenzoic acid-labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N-glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N-glycan peaks from i-QTaG method showed a good linearity (R(2) > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2-AA labeled N-glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up-regulation of the Lewis antigen (~82%) in sera from prostate cancer patients. In this proof-of-concept study, we demonstrated that the i-QTaG method, which enables to achieve a reliable comparative quantitation of total N-glycans via MALDI-TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics.

Proteoglycans in chicken gastrocnemius tendons change with exercise.

Growth, loading, and mobilization lead to changes in tendon structure. Recent studies have shown that proteoglycans (PGs) regulate the organization of collagen fibrils, the main structural components of tendons. We hypothesized that moderate exercise alters PG synthesis in the avian gastrocnemius tendon. To test our hypothesis we compared the PG content in gastrocnemius tendons from control 6.5-week-old chickens with that in tendons from 6.5-week-old chickens that underwent exercise. Our results show high levels and a wide variety of glycosaminoglycans (GAGs) in 6.5-week-old tendons. Chondroitin-4-sulfate disaccharide was the major GAG disaccharide in control and exercised 6.5-week-old gastrocnemius tendons. Exercise led to an increase in the size of the tendons, the content of hyaluronic acid, and the level of decorin. High levels of keratan sulfate (KS) were found in the lower halves of gastrocnemius tendons, although the amount of KS decreased with exercise. This corresponded well with lower content of aggrecan in the lower halves of exercised tendons. In conclusion, our data support the hypothesis that exercise alters the content of PGs in chicken tendons.