RESUMEN
A combination of techniques is presented allowing gel-purified protein identification in the femtomole range using matrix-assisted-laser-desorption-ionization mass spectrometry. The proteins are detected in the primary gel by a sensitive negative staining procedure, transferred, and concentrated in a secondary gel matrix. There, they are digested in the presence of H218O and their sequences are predicted (1) by peptide mass fingerprinting, (2) by comparing the post-source-decay (PSD) spectra with theoretical spectra of candidate isobaric peptides using a computer algorithm called MassFrag, and (3) by a manual readout of the 18O/16O-labeled fragmentation ions in the PSD spectra.
Asunto(s)
Resinas Acrílicas/química , Proteínas/análisis , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Clonación Molecular , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Microquímica/métodos , Datos de Secuencia Molecular , Proteínas/química , Proteínas/aislamiento & purificación , Sensibilidad y Especificidad , Programas Informáticos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrofotometría UltravioletaRESUMEN
FragminP is a Ca2+-dependent actin-binding and microfilament regulatory protein of the gelsolin family. We screened a Physarum polycephalum cDNA library with polyclonal fragminP antibodies and isolated a cDNA clone of 1,104 bp encoding 368 amino acids of fragminP, revealing two consensus phosphatidylinositol 4,5 bisphosphate-binding motifs in the central part of the protein. The first methionine is modified by an acetyl group, and three amino acids were missing from the protein coded for by the cDNA clone. Full-length recombinant fragminP was generated by PCR, purified after over-expression from Escherichia coli and displayed identical properties to native Physarum fragminP. Northern blot analysis against RNA, isolated from cultures at various stages of development, indicated that fragminP is absent from amoebae and that expression is initiated at an early stage during apogamic development, in a similar way to that observed for the profilin genes. In situ immunolocalization of fragminP in Physarum microplasmodia revealed that the protein is localized predominantly at the plasma membrane, suggesting a role in the regulation of the subcortical actin meshwork. Our data indicate that we have isolated the plasmodium-specific fragminP cDNA (frgP) and suggest that, in each of its two vegetative cell types, P. polycephalum uses a different fragmin isoform that performs different functions.