%0 Journal Article
%T Vibratory Reaction Unit for the Rapid Analysis of Proteins and Glycochains
%A Yukie Sasakura
%A Makoto Nogami
%A Noriko Kobayashi and Katsuhiro Kanda
%J Analytical Chemistry Insights
%D 2012
%I 
%X : A protein digestion system using immobilized enzymes for protein identification and glycochain analyses has been developed, and a vibration reaction unit for micro-scale sample convection on an enzyme-immobilized solid surface was constructed. BSA as a model substrate was digested by this unit, and was successfully identified by mass spectrometry (MS) analyses. Compared to the conventional liquid-phase digestion, the reaction unit increased the number of matched peptides from 9 to 26, protein score from 455 to 1247, and sequence coverage from 21% to 48%. Glycopeptidase F (NGF), an enzyme that cleaves N-glycans from glycoproteins, was also immobilized and used to remove the glycochains from human immunoglobulin G (IgG). Trypsin and NGF were immobilized on the same solid surface and used to remove glycochains from IgG in single-step. Glycochains were labeled with fluorescent reagent and analyzed by HPLC. Several peaks corresponding to the glycochains of IgG were detected. These results suggested that the single-step digestion system, by immobilized multiple enzymes (trypsin and NGF) would be effective for the rapid structural analysis of glycoproteins. Abbreviations: BSA: bovine serum albumin; MS: mass spectrometry; NGF: glycopeptidase F; IgG: immunoglobulin G; PTM: post-translational modification; HPLC: high-performance liquid chromatography; PBS: phosphate-buffered saline; EDTA: ethylenediaminetetraacetic acid; DTT: dithiothreitol; RT: retention time; ABOE: p-aminobenzoic acid octyl ester; PDMS: polydimethylsiloxane; ArgC: endoprotease Arginine C.
%U http://www.la-press.com/vibratory-reaction-unit-for-the-rapid-analysis-of-proteins-and-glycoch-article-a361