Protein Surface Mapping Using Diethylpyrocarbonate with Mass Spectrometric Detection

Abstract

The reliability and information content of diethylpyrocarbonate (DEPC) as a covalent probe of protein surface structure has been improved when used appropriately with mass spectrometric detection. Using myoglobin, cytochrome c, and β-2-microglobulin as model protein systems, we demonstrate for the first time that DEPC can modify Ser and Thr residues in addition to His and Tyr residues. This result expands the capability of DEPC as a structural probe because about 25% of the sequence of the average protein can now be covered using this covalent labeling reagent. In addition, we establish a new approach based on mass spectrometry to ensure the structural integrity of proteins during amino acid-specific covalent labeling reactions. This approach involves monitoring the extent of modification as a function of reagent concentration and allows any small-scale or local perturbations caused by the covalent label to be readily identified and avoided. Results indicate that these dose-response plots are much more reliable and generally applicable probes of possible protein structural changes than fluorescence or circular dichroism spectroscopies. These dose-response plots also provide a means of quantitatively comparing the reactivity of each modified residue. Based on comparisons to known X-ray crystal structures, we find that the solvent accessibility of the reactive atom in the side chain and the presence of a nearby charged residue most affect modification rates. Finally, this improved surface mapping method has been used to determine the effect of Cu(II) binding on the structure of β-2-microglobulin. Results confirm that Cu(II) binds His31, but not any of the other three His residues, and changes the solvent accessibility of residues near His31 and near the N-terminus.

Characterizing monoclonal antibody structure by carbodiimide/GEE footprinting

“…The chemistry of these reagents varies such that a wide range of amino acid side chains can be probed providing structure assessment of proteins with single side chain resolution. [31][32][33][34][35][36][37][38][39] In particular, footprinting can be performed with either less-specific labels, such as hydroxyl radicals that can label more than 16 side chains or more specific labels that target a few particular residues. Because understanding how changes in formulation, alterations in post-translational modifications such as glycosylation, or other changes in the production of the protein influence the structure and thus its activity is crucial when developing therapeutic proteins, CL-based footprinting methods can provide quantitative assessments of tertiary and quaternary structure.…”

Protein-metal interactions of calmodulin and α-synuclein monitored by selective noncovalent adduct protein probing mass spectrometry

“…For example, Gross et al found that the number of exchangeable hydrogens decreases as the concentration of Ca 2ϩ is increased up to ϳ0.25 mM, which they interpret as a tightening of the Ca 2ϩ binding domains of calmodulin [43,44]. Reactive labeling groups can also be used to monitor protein surface structure [45]. Specific, noncovalent interactions with the side-chains of proteins can also be used to probe protein structure [46 -50].…”

Mass spectrometry-enabled structural biology of membrane proteins

“…to probe Arg accessibility, and has been used to study the conformational transitions in LacY, in combination with diisopropyl carbodiimide labeling of carboxyl groups [241]. Diethylpyrocarbonate (DEPC) is also a commonly encountered modifying reagent, and whilst it primarily targets His it also has the potential to react with Lys, Arg, Tyr, Thr and Cys residues [242,243]. DEPC, along with all other covalent labels, is able to provide restraints for integrative modeling workflows, as exemplified in recent work on the F 0 F 1 ATPase from spinach chloroplasts (in combination with XL-MS data) [243].…”