Mass spectrometry tools for analysis of intermolecular interactions

Abstract

The small quantities of protein required for mass spectrometry (MS) make it a powerful tool to detect binding (protein-protein, protein-small molecule, etc.) of proteins that are difficult to express in large quantities, as is the case for many intrinsically disordered proteins. Chemical cross-linking, proteolysis, and MS analysis, combined, are a powerful tool for the identification of binding domains. Here, we present a traditional approach to determine protein-protein interaction binding sites using heavy water ((18)O) as a label. This technique is relatively inexpensive and can be performed on any mass spectrometer without specialized software.

Fig. 1

Mass spectrometry analysis of protein binding work-flow.

Fig. 2

Heavy water labeled peptides and cross-linked peptides. (a) Mass spectra of a peptide; box zoomed in (b). (b) An unlabelled m/z at 1,380.7 and its labeled counterpart at 1,384.7; the shift in mass of four indicates a non-cross-linked peptide. (c) Mass Spectra of a cross-linked peptide (box; zoomed in (d)). Analyzing the spectra by hand allows for the majority of cross-linked pairs to be identified. (d) An unlabelled m/z at 2,156.1 and its labeled counterpart at 2,164.5; the shift in mass of eight indicates a cross-linked peptide. The cross-linking complicates the spectral analysis; however, it is still possible to distinguish the pair of related m/z values based on the isotopic label.

Fig. 3

Noncovalent Interactions using the FTICR. (Top) Spectra of protein alone. (Bottom) Protein incubated with a panel of small molecules. The addition of a new peak indicates protein–compound complex (noncovalent interaction).