Journal
ANALYTICAL CHEMISTRY
Volume 83, Issue 17, Pages 6738-6745Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac201376q
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Funding
- NIH/NCSU [5T32GM00-8776-08]
- W. M. Keck Foundation
- North Carolina State University
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This study presents the development of stable-isotope labeled hydrophobic, hydrazide reagents for the relative quantification of N-linked glycans. The P2GPN light (C-12) and heavy (C-13(6)) pair are used to differentially label two N-linked glycan samples. The samples are combined 1:1, separated using HILIC, and then mass differentiated and quantified using mass spectrometry. These reagents have several benefits: (1) impart hydrophobic character to the glycans affording an increase in electrospray ionization efficiency and MS detection; (2) indistinguishable chromatographic, MS, and MS/MS performance of the light and heavy reagents affording relative quantification; and (3) analytical variability is significantly reduced due to the two samples being mixed together after sample preparation. Obtaining these analytical benefits only requires similar to 4 h of sample preparation time. It is shown that these reagents are capable of quantifying changes in glycosylation in simple mixtures, and the analytical variability of the reagents in pooled plasma samples is shown to be less than +/- 30%. Additionally, the incorporation of an internal standard allows one to account for the difference in systematic error between the two samples due to the samples being processed in parallel and not mixed until after derivatization.
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