Twoplex p>12/13p>C₆ aniline stable isotope and linkage-specific sialic acid labeling 2D-LC-MS workflow for quantitative N-glycomics

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• 作者：Simone Albrecht ; Stefan Mittermayr ; Josh Smith ; Silvia Mill&aacute ; n Martí ; n ; Margaret Doherty and Jonathan Bones
• 刊名：PROTEOMICS
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：17
• 期：1-2
• 全文大小：1558K
• ISSN：1615-9861

文摘

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-MSp>Ep> workflow for the fractionation and relative quantitation of twoplex isotopically labeled N-linked oligosaccharides using neutral p>12p>C₆ and p>13p>C₆ aniline (Δmass = 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 p>12p>C₆/p>13p>C₆ 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.