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  • 标题:High-throughput analysis of N-glycans using AutoTip via glycoprotein immobilization
  • 本地全文:下载
  • 作者:Shuang Yang ; David Clark ; Yang Liu
  • 期刊名称:Scientific Reports
  • 电子版ISSN:2045-2322
  • 出版年度:2017
  • 卷号:7
  • 期号:1
  • DOI:10.1038/s41598-017-10487-8
  • 语种:English
  • 出版社:Springer Nature
  • 摘要:Analysis of a large number of samples requires an efficient, rapid and reproducible method. Automation is an ideal approach for high-throughput sample preparation. Multi-plexing sample preparation via a 96-well plate format becomes popular in recent years; however, those methods lack specificity and require several cleanup steps via chromatography purification. To overcome these drawbacks, a chemoenzymatic method has been developed utilizing protein conjugation on solid-phase. Previously, sample preparation was successfully performed in a snap-cap spin-column (SCSC) format. However, sample preparation using SCSC is time-consuming and lacks reproducibility. In this work, we integrated the chemoenzymatic technique in a pipette tip (AutoTip) that was operated by an automated liquid handler. We established a multi-step protocol involving protein immobilization, sialic acid modification, and N-glycan release. We first optimized our automated protocol using bovine fetuin as a standard glycoprotein, and then assessed the reproducibility of the AutoTip using isobaric tags for relative N-linked glycan quantification. We then applied this methodology to profile N-glycans from 58 prostate cancer patient urine samples, revealing increased sialyation on urinary N-glycans derived from prostate cancer patients. Our results indicated AutoTip has applications for high-throughput sample preparation for studying the N-linked glycans.
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