使用液相色谱-质谱联用技术对肌醇六磷酸酶促制备的去磷酸化产物进行分离和鉴定。

Separation and identification of enzymatically prepared dephosphorylated products of myo-inositolhexakisphosphate using LC-MS.

作者信息

Vats Purva, Bhushan Bharat, Chakarborti Asit K, Banerjee Uttam C

机构信息

Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, USA.

出版信息

J Sep Sci. 2008 Dec;31(22):3829-33. doi: 10.1002/jssc.200800372.

DOI:10.1002/jssc.200800372

PMID:19009537

Abstract

LC-MS technique described here is a new way for the separation and direct determination of UV-Vis insensitive inositol phosphates (InsP(2)-InsP(6)). This circumvents the need of radioisotopic labeling and post-column derivatization techniques. The method involves separation of various enzymatically dephosphorylated derivatives of InsP(6) on C(18)-column using MeOH/H(2)O (30:70 v/v) and their identification using electron spray ionization MS in positive ion mode (+pESI-MS). The LC-MS studies revealed that the purified phytase from Aspergillus niger van Teighem hydrolyzes InsP(6 )in a sequential manner leading to InsP(2 )(InsP(2) x 2Na, t(R) 4.4-4.54 min, base peak m/z 382.9) as the end product.

摘要

本文所述的液相色谱-质谱联用（LC-MS）技术是分离和直接测定对紫外-可见光谱不敏感的肌醇磷酸酯（InsP(2)-InsP(6)）的一种新方法。这避免了放射性同位素标记和柱后衍生技术的需求。该方法包括使用甲醇/水（30:70 v/v）在C(18)柱上分离InsP(6)的各种酶促去磷酸化衍生物，并使用正离子模式的电喷雾电离质谱（+pESI-MS）对其进行鉴定。LC-MS研究表明，黑曲霉范特伊格姆纯化的植酸酶以连续方式水解InsP(6)，最终产物为InsP(2)（InsP(2) x 2Na，保留时间4.4 - 4.54分钟，基峰m/z 382.9）。