Open AccessCapillary Zone Electrophoresis-Electron-Capture Collision-Induced Dissociation on a Quadrupole Time-of-Flight Mass Spectrometer for Top-Down Characterization of Intact Proteins
Author(s) -
Xiaojing Shen,
Tian Xu,
Blake Hakkila,
Michael Hare,
Qianjie Wang,
Qianyi Wang,
Joseph S. Beckman,
Liangliang Sun
Publication year - 2021
Publication title -
journal of the american society for mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.961
H-Index - 127
eISSN - 1879-1123
pISSN - 1044-0305
DOI - 10.1021/jasms.0c00484
Subject(s) - chemistry , electron transfer dissociation , orbitrap , mass spectrometry , electron capture dissociation , fragmentation (computing) , top down proteomics , capillary electrophoresis , collision induced dissociation , dissociation (chemistry) , analytical chemistry (journal) , chromatography , quadrupole ion trap , tandem mass spectrometry , selected reaction monitoring , ion trap , computer science , operating system
Mass spectrometry (MS)-based denaturing top-down proteomics (dTDP) requires high-capacity separation and extensive gas-phase fragmentation of proteoforms. Herein, we coupled capillary zone electrophoresis (CZE) to electron-capture collision-induced dissociation (ECciD) on an Agilent 6545 XT quadrupole time-of-flight (Q-TOF) mass spectrometer for dTDP for the first time. During ECciD, the protein ions were first fragmented using ECD, followed by further activation and fragmentation by applying a CID potential. In this pilot study, we optimized the CZE-ECciD method for small proteins (lower than 20 kDa) regarding the charge state of protein parent ions for fragmentation and the CID potential applied to maximize the protein backbone cleavage coverage and the number of sequence-informative fragment ions. The CZE-ECciD Q-TOF platform provided extensive backbone cleavage coverage for three standard proteins lower than 20 kDa from only single charge states in a single CZE-MS/MS run in the targeted MS/MS mode, including ubiquitin (97%, +7, 8.6 kDa), superoxide dismutase (SOD, 87%, +17, 16 kDa), and myoglobin (90%, +16, 17 kDa). The CZE-ECciD method produced comparable cleavage coverage of small proteins (i.e., myoglobin) with direct-infusion MS studies using electron transfer dissociation (ETD), activated ion-ETD, and combinations of ETD and collision-based fragmentation on high-end orbitrap mass spectrometers. The results render CZE-ECciD a new tool for dTDP to enhance both separation and gas-phase fragmentation of proteoforms.