変異蛋白，翻訳後修飾蛋白のＭＳによる構造解析

Abstract

Progress in medicine and molecular research has been increased by the need for simple and reliable methods of detection and characterization of aberrant proteins caused either by mutation or by post-translational modification. We applied electrospray ionization mass spectrometry (ESIMS) to detect and characterize abnormal structure proteins, and to determine the ratio of mutant and wild type proteins. Direct examination of hemolysate might well lead to rapid ascertainment of variant hemoglobins (Hbs) provided that the mass difference between the normal and the abnormal chain is larger than the resolution power of standard instruments. Various procedures are required in the presentation of most plasma and cell proteins other than Hb prior to MS. Two simple methods were deviced to prepare proteins. One was immunoprecipitation with antisera against target proteins, followed by reversed-phase HPLC/ESIMS, and the other was 2-dimensional LC (a strong anion exchange region and a reversed-phase resin) connected to ESIMS. With both methods, the ions of intact normal and variant proteins were clearly observed in samples from patients with neurodegenerative diseases such as familial amyloidotic polyneuropathy (FAP) and amyotrophic lateral screlosis (FALS). Using these procedures, we detected more than 40 cases of transthyretins (TTRs) mutants including to 10 different types. Two of these, [101 Gly->Ser] and [38 Asp->Ala], were new. We also successfully detected 4 different mutants of Cu/Zn-binding superoxide dismutase inerythrocytes, and a variant in spinal cord from patients with FALS by immunoprecipitation mehods. Two of these, [4Ala->Ser] and [111Cys->Tyr], were new. Finally we also measured glycated β-globin N-ternimus hexapeptide (HbA1c) by Poroszyme V8 protease digestion and nanoESIMS technique.