Probing Protein 3D Structures and Conformational Changes Using Electrochemistry-Assisted Isotope Labeling Cross-Linking Mass Spectrometry

Qiuling Zheng, Hao Zhang, Shiyong Wu, Hao Chen

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

This study presents a new chemical cross-linking mass spectrometry (MS) method in combination with electrochemistry and isotope labeling strategy for probing both protein three-dimensional (3D) structures and conformational changes. For the former purpose, the target protein/protein complex is cross-linked with equal mole of premixed light and heavy isotope labeled cross-linkers carrying electrochemically reducible disulfide bonds (i.e., DSP-d0 and DSP-d8 in this study, DSP = dithiobis[succinimidyl propionate]), digested and then electrochemically reduced followed with online MS analysis. Cross-links can be quickly identified because of their reduced intensities upon electrolysis and the presence of doublet isotopic peak characteristics. In addition, electroreduction converts cross-links into linear peptides, facilitating MS/MS analysis to gain increased information about their sequences and modification sites. For the latter purpose of probing protein conformational changes, an altered procedure is adopted, in which the protein in two different conformations is cross-linked using DSP-d0 and DSP-d8 separately, and then the two protein samples are mixed in 1:1 molar ratio. The merged sample is subjected to digestion and electrochemical mass spectrometric analysis. In such a comparative cross-linking experiment, cross-links could still be rapidly recognized based on their responses to electrolysis. More importantly, the ion intensity ratios of light and heavy isotope labeled cross-links reveal the conformational changes of the protein, as exemplified by examining the effect of Ca2+ on calmodulin conformation alternation. This new cross-linking MS method is fast and would have high value in structural biology. [Figure not available: see fulltext.]

Original languageEnglish (US)
Pages (from-to)864-875
Number of pages12
JournalJournal of the American Society for Mass Spectrometry
Volume27
Issue number5
DOIs
StatePublished - May 1 2016
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Spectroscopy

Keywords

  • Chemical cross-linking
  • Electrochemistry
  • Isotope-labeling
  • Mass spectrometry
  • Protein 3D structure
  • Protein conformational change

Fingerprint

Dive into the research topics of 'Probing Protein 3D Structures and Conformational Changes Using Electrochemistry-Assisted Isotope Labeling Cross-Linking Mass Spectrometry'. Together they form a unique fingerprint.

Cite this