使用反相高效液相色谱法分离和分析α-麦醇溶蛋白的肽片段

Isolation and analysis of peptidic fragments of alpha-gliadin using reversed-phase high-performance liquid chromatography.

作者信息

Kocna P, Fric P, Kocová-Holáková M, Slabý J, Kasafírek E, Hekkens W T

机构信息

Laboratory of Gastroenterology, Charles University, Faculty of General Medicine, Prague, Czechoslovakia.

出版信息

J Chromatogr. 1988 Dec 30;434(2):429-38. doi: 10.1016/s0378-4347(88)80010-0.

DOI:10.1016/s0378-4347(88)80010-0

PMID:3246533

Abstract

Peptidic fragments of alpha-gliadin were obtained by peptic-tryptic-pancreatic (PTP) digestion of the alpha-gliadin fraction isolated by ion-exchange chromatography on a sulphopropyl-Sephadex C-50 column. The proteolytic digest was fractionated by ultrafiltration into three subfractions, PTPa1-PTPa3. The subfraction PTPa2 was then analysed and individual peaks were separated using reversed-phase high-performance liquid chromatography (RP-HPLC) using a gradient of acetonitrile in 0.1% trifluoroacetic acid and a Separon SGX-C18 sorbent. A 100-mg amount of the PTPa2 subfraction was separated in a single analysis by preparative RP-HPLC and twenty peaks were obtained for further characterization. The molecular mass in range 300-3000 was established for individual peptidic fragments by gel-permeation chromatography on a TSK-G2000 SW column.

摘要

通过在磺丙基 - 葡聚糖凝胶C - 50柱上进行离子交换色谱分离得到α - 麦醇溶蛋白组分，再经胃蛋白酶 - 胰蛋白酶 - 胰酶（PTP）消化获得α - 麦醇溶蛋白的肽片段。蛋白水解消化物通过超滤分离为三个亚组分，即PTPa1 - PTPa3。然后对亚组分PTPa2进行分析，并使用乙腈在0.1%三氟乙酸中的梯度以及Separon SGX - C18吸附剂，通过反相高效液相色谱（RP - HPLC）分离各个峰。通过制备型RP - HPLC在单次分析中分离出100 mg的PTPa2亚组分，并获得20个峰用于进一步表征。通过在TSK - G2000 SW柱上进行凝胶渗透色谱法确定各个肽片段的分子量范围为300 - 3000。