Mass spectrometry-based carboxyl footprinting of proteins: Method evaluation

Protein structure determines function in biology, and a variety of approaches have been employed to obtain structural information about proteins. Mass spectrometry-based protein footprinting is one fast-growing approach. One labeling-based footprinting approach is the use of a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and glycine ethyl ester (GEE) to modify solvent-accessible carboxyl groups on glutamate (E) and aspartate (D). This paper describes method development of carboxyl-group modification in protein footprinting. The modification protocol was evaluated by using the protein calmodulin as a model. Because carboxyl-group modification is a slow reaction relative to protein folding and unfolding, there is an issue that modifications at certain sites may induce protein unfolding and lead to additional modification at sites that are not solvent-accessible in the wild-type protein. We investigated this possibility by using hydrogen deuterium amide exchange (H/DX). The study demonstrated that application of carboxyl group modification in probing conformational changes in calmodulin induced by Ca2+ binding provides useful information that is not compromised by modification-induced protein unfolding.

Graphical abstract.Figure optionsDownload full-size imageDownload high-quality image (109 K)Download as PowerPoint slideHighlights► Carboxyl-group modification is proposed as footprint of proteins in bio settings. ► Carboxyl footprinting responds to conformation in regions with these side chains. ► This footprinting induces no major conformational change or over-labels protein. ► H/D amide exchange is a means to insure footprinting does not perturb conformation.