Probing protein surface with a solvent mimetic carbene coupled to detection by mass spectrometry

GABRIELA E. GÓMEZ; MARIANA R. MUNDO; PATRICIO O. CRAIG; JOSÉ M. DELFINO

JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY

ELSEVIER SCIENCE INC

Año: 2012 vol. 23 p. 30 - 42

1044-0305

Much knowledge into protein folding, ligand binding, and complex formation can be derived fromthe examination of the nature and size of the accessible surface area (SASA) of the polypeptidechain, a key parameter in protein science not directly measurable in an experimental fashion. Tothis end, an ideal chemical approach should aim at exerting solvent mimicry and achievingminimal selectivity to probe the protein surface regardless of its chemical nature. The choice ofthe photoreagent diazirine to fulfill these goals arises from its size comparable to water and frombeing a convenient source of the extremely reactive methylene carbene (:CH2). The ensuingmethylation depends primarily on the solvent accessibility of the polypeptide chain, turning it intoa valuable signal to address experimentally the measurement of SASA in proteins. The superbsensitivity and high resolution of modern mass spectrometry techniques allows us to derive aquantitative signal proportional to the extent of modification (EM) of the sample. Thus, diazirinelabeling coupled to electrospray mass spectrometry (ESI-MS) detection can shed light onconformational features of the native as well as non-native states, not easily addressable byother methods. Enzymatic fragmentation of the polypeptide chain at the level of small peptidesallows us to locate the covalent tag along the amino acid sequence, therefore enabling theconstruction of a map of solvent accessibility. Moreover, by subsequent MS/MS analysis ofpeptides, we demonstrate here the feasibility of attaining amino acid resolution in defining thetarget sites.