Correlation between Labeling Yield and Surface Accessibility in Covalent Labeling Mass Spectrometry

Abstract

The functional properties of a protein are strongly influenced by its topography, or the solvent-facing contour map of its surface. Together with crosslinking, covalent labeling mass spectrometry (CL-MS) has the potential to contribute topographical data through the measurement of surface accessibility. However, recent efforts to correlate measures of surface accessibility with labeling yield have been met with mixed success. Most applications of CL-MS involve differential analysis of protein interactions (i.e., footprinting experiments) where such inconsistencies have limited effect. Extending CL-MS into structural analysis requires an improved evaluation of the relationship between labeling and surface exposure. In this study, we applied recently developed diazirine reagents to obtain deep coverage of the large motor domain of Eg5 (a mitotic kinesin), and together with computational methods we correlated labeling yields with accessibility data in a number of ways. We observe that correlations can indeed be seen at a local structural level, but these correlations do not extend across the structure. The lack of correlation arises from the influence of protein dynamics and chemical composition on reagent partitioning and, thus, also on labeling yield. We conclude that our use of CL-MS data should be considered in light of “chemical accessibility” rather than “solvent accessibility” and suggest that CL-MS data would be a useful tool in the fundamental study of protein–solute interactions.

Residue-Level Characterization of Antibody Binding Epitopes Using Carbene Chemical Footprinting

“…This residue-level surface coverage also produces one of the highest levels of structural resolution compared to results obtained by other chemical footprinting techniques . Structure penetration by the two reagents was observed with 26.8% of the TDBA- and 11.1% of the 3-azibutanol-labeled residues having SASA values <0.2, similar to another study . Interestingly, the TDBA reagent produced greater surface coverage of the α1 domain, whereas regions of the α2 domain were better covered by the 3-azibutanol reagent, which is directly dependent on the identity and location of the surface-exposed amino acids given the reactivity of each reagent for various amino acids, although the local microenvironment may also be a factor.…”

Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications

“…145,1220 Understanding the effect of the microenvironment on labeling efficiencies is crucial in determining the intrinsic structural factors that are involved in interpreting footprinting data, and these effects should not be overlooked. Some recent reports suggest that the extent of labeling by irreversible labeling reagents are not strictly driven by SASA, but by "chemical accessibility" that are a function of local or microenvironments 143,144 (the idea of "chemical accessibility" was mentioned in section 1.3.1). Only from a deep understanding can we take full advantage of footprinting results and their incorporation into protein structural modeling.…”

“…There are examples of HDX where protection does not increase at a binding interface . Other examples indicate that differences are determined by “chemical accessibility” rather than surface accessibility. , There are also examples of radical precursors that interact by H-bonding and give a footprint that is disproportionate to accessible surface, challenging the simple notion that a reactivity only depends on surface area . Although these scenarios indicate more complex factors behind the apparent modification extent, the labeling process can still be well represented by the term “footprinting”.…”

Atomistic Details of Peptide Reversed-Phase Liquid Chromatography from Molecular Dynamics Simulations

“…RPLC/ESI-MS is particularly important for proteomics. Most bottom-up workflows involve the analysis of peptides generated using trypsin, a protease that cleaves on the C-terminal side after Arg and Lys (unless followed by Pro). − Similar RPLC-based bottom-up strategies are used for protein hydrogen/deuterium exchange, covalent labeling, , and cross-linking. , Peptide separation in bottom-up experiments is typically performed on C18 columns with a water/acetonitrile LC gradient in the presence of 0.1% formic acid. ,,,,,, Under these low pH conditions, acidic groups (Asp and Glu side chains, C-termini) are neutral, while basic sites (Arg, Lys, His, N-termini) are positively charged…”

“…Most bottom-up workflows involve the analysis of peptides generated using trypsin, a protease that cleaves on the C-terminal side after Arg and Lys (unless followed by Pro). 24−26 Similar RPLC-based bottom-up strategies are used for protein hydrogen/deuterium exchange, 27 covalent labeling, 28,29 and cross-linking. 30,31 Peptide separation in bottom-up experiments is typically performed on C18 columns with a water/acetonitrile LC gradient in the presence of 0.1% formic acid.…”