Probing Protein Surface with a Solvent Mimetic Carbene Coupled to Detection by Mass Spectrometry

Abstract

Much knowledge into protein folding, ligand binding, and complex formation can be derived from the examination of the nature and size of the accessible surface area (SASA) of the polypeptide chain, a key parameter in protein science not directly measurable in an experimental fashion. To this end, an ideal chemical approach should aim at exerting solvent mimicry and achieving minimal selectivity to probe the protein surface regardless of its chemical nature. The choice of the photoreagent diazirine to fulfill these goals arises from its size comparable to water and from being a convenient source of the extremely reactive methylene carbene (:CH 2 ). The ensuing methylation depends primarily on the solvent accessibility of the polypeptide chain, turning it into a valuable signal to address experimentally the measurement of SASA in proteins. The superb sensitivity and high resolution of modern mass spectrometry techniques allows us to derive a quantitative signal proportional to the extent of modification (EM) of the sample. Thus, diazirine labeling coupled to electrospray mass spectrometry (ESI-MS) detection can shed light on conformational features of the native as well as non-native states, not easily addressable by other methods. Enzymatic fragmentation of the polypeptide chain at the level of small peptides allows us to locate the covalent tag along the amino acid sequence, therefore enabling the construction of a map of solvent accessibility. Moreover, by subsequent MS/MS analysis of peptides, we demonstrate here the feasibility of attaining amino acid resolution in defining the target sites.

Evolution of Structural Biology through the Lens of Mass Spectrometry

“…A recently developed nonspecific covalent label is the use of carbenes that are photoreactive. 104 The addition of diazirines to the solution followed by irradiation at near-ultraviolet wavelength (310–350 nm), which is outside the absorbance window of amino acids, results in the formation of reactive carbenes. Once formed, the carbene has an approximate lifetime of a few ns, which allows fast sampling of a protein state.…”

“…The advantage of using a specific covalent label is that it reduces the complexity of data analysis. A recently developed nonspecific covalent label is the use of carbenes that are photoreactive . The addition of diazirines to the solution followed by irradiation at near-ultraviolet wavelength (310–350 nm), which is outside the absorbance window of amino acids, results in the formation of reactive carbenes.…”

Structural analysis of a therapeutic monoclonal antibody dimer by hydroxyl radical footprinting

“…Various labels can be utilized, including amino acid side chain-specific labeling reagents such as iodoacetamide (cysteines), N-bromosuccinimide (tryptophan), diethylpyrocarbonate (carboxyl acids), and S,S 1 -dimethylthiobutanoy lhdroxysuccinimide (lysines). [14][15][16][17] Structural information from these reagents may not be as comprehensive due to their inherent specificities.…”

Protein Footprinting by Carbenes on a Fast Photochemical Oxidation of Proteins (FPOP) Platform

“…Delfino et al [17, 18] used 3 H-methylene so that its concentration in solution can be monitored, and they demonstrated that methylene reactivity is a monitor of protein conformational transitions and protein interactions. In their later work, Delfino et al [19, 20] used mass spectrometry as the analytical method to achieve accurate detection of footprinting products and map solvent accessibility of proteins. Gas bubbling devices were used in their experiments to mix methylene gas with protein solutions, and reactions were initiated by a halogen/Hg lamp or a Hg/Xe arc source.…”