INVESTIGADORES
DELFINO Jose Maria
artículos
Título:
Probing Protein Surface with a Solvent Mimetic Carbene Coupled to Detection by Mass Spectrometry
Autor/es:
GÓMEZ GE; MUNDO MR; CRAIG PO; DELFINO JM
Revista:
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
Editorial:
ELSEVIER SCIENCE INC
Referencias:
Lugar: Amsterdam; Año: 2011 vol. 23 p. 30 - 42
ISSN:
1044-0305
Resumen:
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 (:CH2). 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.