Evaluation of NHS-Acetate and DEPC labelling for determination of solvent accessible amino acid residues in protein complexes.

The activity of most proteins and protein complexes relies on the formation of defined three-dimensional structures. The analysis of these arrangements is therefore key for understanding their function and regulation in the cell. Besides the traditional structural techniques, structural mass spectrometry delivers insights into the various aspects of protein structure, including stoichiometry, protein-ligand interactions and solvent accessibility. The latter is usually obtained from labelling experiments. In this study, we evaluate two chemical labelling strategies using N-hydroxysuccinimidyl acetate and diethylpyrocarbonate as labelling reagents. We characterised the mass spectra of modified peptides and assessed labelling reactivity of individual amino acid residues in intact proteins. Importantly, we uncovered neutral losses from diethylpyrocarbonate modified amino acids improving the assignments of the peptide fragment spectra. We further established a quantitative labelling workflow to determine labelling percentage and unambiguously distinguish solvent accessible amino acid residues from stochastically labelled residues. Finally, we used ion mobility MS to explore whether labelled proteins maintain their structures and remain stable. We conclude that labelling using N-hydroxysuccinimidyl acetate and diethylpyrocarbonate delivers comparable results, however, N-hydroxysuccinimidyl acetate labelling is compatible with standard proteomic workflows while diethylpyrocarbonate labelling requires specialised experimental conditions and data analysis. SIGNIFICANCE: Covalent labelling is widely used to identify solvent accessible amino acid residues of proteins or protein complexes. However, with increasing sensitivity of available MS instrumentation, a high number of modified residues is usually observed making an unambiguous assignment of solvent accessible residues necessary. In this study, we establish a quantitative labelling workflow for two different labelling strategies to identify accessible amino acid residues. In addition, we characterise observed mass spectra of modified peptides and identified neutral loss of DEPC modified amino acid residues during HCD fragmentation improving their assignments.