Development of an online microbore hollow fiber enzyme reactor coupled with nanoflow liquid chromatography-tandem mass spectrometry for global proteomics

Jin Yong Kim, Sun Young Lee, Sook Kyung Kim, Sang Ryoul Park, Dukjin Kang, Myeong Hee Moon

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

In this study, we report the development of a microbore hollow fiber enzyme reactor (mHFER) coupled to nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) for the online digestion or selective enrichment of glycopeptides and analysis of proteins. With mHFER, enzymatic digestion of protein could be achieved by continuous flow within a very small volume (∼10 μL) of mHF inserted in a PEEK tube. Digested peptides exited through the pores of the hollow fiber membrane wall to external single or multiplexed trap columns for nLC-ESI-MS/MS analysis. Evaluation of online mHFER-nLC-ESI-MS/MS system was made with bovine serum albumin (BSA) by varying the temperature of digestion and the amount of protein injected. We evaluated the ability of the mHFER system to enrich glycopeptides by injecting a mixture of lectin (concanavalin A) and digested peptides from α-1-acid glycoprotein (AGP) into the mHFER, followed by delivery of PNGase F for endoglycosidic digestion. Nonglycosylated peptides unbound to lectins eluted at the first breakthrough run while N-linked glycopeptides eluted after the endoglycosidic digestion. The developed method was applied to urine samples from patients with prostate cancer and controls; 67 N-linked glycopeptides were identified and relative differences in glycopeptide content between patient and control samples were determined.

Original languageEnglish
Pages (from-to)5506-5513
Number of pages8
JournalAnalytical Chemistry
Volume85
Issue number11
DOIs
Publication statusPublished - 2013 Jun 4

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All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

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