Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies

J Am Soc Mass Spectrom. 2016 Jan;27(1):178-81. doi: 10.1007/s13361-015-1260-5. Epub 2015 Sep 18.

Abstract

We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded "heavy" and "light" GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the "heavy" and "light" peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting.

Keywords: Cyanobacteria; Glycine ethyl ester (GEE); Isotope encoding; Mass spectrometry; Orange carotenoid protein; Protein footprinting.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins / analysis
  • Bacterial Proteins / chemistry
  • Chromatography, Liquid
  • Glycine / analogs & derivatives
  • Glycine / chemistry
  • Isotope Labeling / methods*
  • Models, Molecular
  • Peptide Fragments / analysis*
  • Peptide Fragments / chemistry*
  • Protein Conformation
  • Protein Footprinting / methods*
  • Tandem Mass Spectrometry / methods*

Substances

  • Bacterial Proteins
  • Peptide Fragments
  • orange carotenoid protein, Synechocystis
  • glycine ethyl ester
  • Glycine