Journal
PROTEOMICS
Volume 17, Issue 1-2, Pages -Publisher
WILEY
DOI: 10.1002/pmic.201600304
Keywords
DMT-MM; Glycan quantitation; Glycoproteomics; LC-MS; Sialic acid; Stable isotope aniline
Funding
- EU [FP7-PEOPLE-316929, FP7-HEALTH-259869]
- Science Foundation Ireland [11/SIRG/B2107, 08/SRC/B1393]
- Science Foundation Ireland (SFI) [11/SIRG/B2107] Funding Source: Science Foundation Ireland (SFI)
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Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease-associated glycan alterations to the quantitative characterization of N-glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI-TOF-MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run-to-run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC-MSE workflow for the fractionation and relative quantitation of twoplex isotopically labeled N-linked oligosaccharides using neutral C-12(6) and C-13(6) aniline (Delta mass = 6 Da). Additional linkage-specific derivatization of sialic acids using 4-(4,6-dimethoxy-1,3,5-trizain-2-yl)-4-methylmorpholinium chloride offered simultaneous and advanced in-depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N-glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex C-12(6)/C-13(6) aniline 2D LC-MS platform is ideally suited for differential glycomic analysis of structurally complex N-glycan pools due to combination and analysis of samples in a single LC-MS injection and the associated minimization in technical variation.
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