Limited proteolysis combined with isotope labeling and quantitative LC-MALDI MS for monitoring protein conformational changes: a study on calcium-binding sites of cardiac Troponin C |
| |
Authors: | Chris McDonald |
| |
Affiliation: | Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 |
| |
Abstract: | Studies of protein-protein and protein-ligand interactions are important for understanding biological functions of proteins. A new technique based on the partial proteolysis of proteins combined with quantitative mass spectrometry is developed as a means of tracking structural changes after the formation of a protein-ligand complex. In this technique, a protein of interest with and without the binding of a ligand is digested with an enzyme to generate a set of peptides, followed by separation of the peptides by liquid chromatography. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is used to identify chromatographically separated peptides, and locate their sequence alignments in the parent protein. Using an isotopically labeled protein as a sample against an unlabeled protein standard, quantitative information can be gathered. This overcomes the inherent lack of quantitative capability of MALDI MS. The utility of the technique to investigate protein-ligand interactions is demonstrated in a model system involving calcium binding to cardiac Troponin C (cTnC). Using this technique, the general location of the three calcium-binding sites of cTnC can be determined by using several different enzymes to generate overlapping peptide maps of cTnC. |
| |
Keywords: | Liquid chromatography Matrix-assisted laser desorption ionization mass spectrometry Cardiac Troponin C Quantitative mass spectrometry |
本文献已被 ScienceDirect 等数据库收录! |
|