RESUMEN
Mass spectrometry is a powerful technique for the identification of proteins at nanogram quantities. However, some degree of sample preparation prior to mass spectrometry is required, and silver-stained protein gel samples are most problematic. Here we report our strategy to obtain peptide mass profiles from silver-stained protein gel samples from one- or two-dimensional gels by destaining prior to enzymatic digestion. This study demonstrates that by using the destaining method, the sensitivity and quality of mass spectra is increased for matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis, permitting more proteins to be identified by peptide mass database analysis.
Asunto(s)
Resinas Acrílicas , Proteínas/análisis , Tinción con Nitrato de Plata/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Electroforesis en Gel Bidimensional/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Iones , Sensibilidad y EspecificidadRESUMEN
As the resource laboratory for Rockefeller University our emphasis continues to be on methodology development for the routine analysis of low abundance proteins isolated from native sources. In the past ten years, gel electrophoresis of proteins has become the method of choice for the preparation of microgram and submicrogram quantities of protein for primary structural characterization, and over 95% of the samples submitted for protein identification are either in a gel or bound to polyvinyl difluoride membranes (PVDF). As such, we employ multiple microanalytical approaches encompassing Edman sequence degradation, amino acid and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis to provide an integrated protein characterization of such samples. Here we describe the two major services we employ when providing protein identification from in-gel or PVDF-bound proteins.
Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Membranas Artificiales , Mapeo Peptídico/métodos , Polivinilos , Proteínas/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Hidrólisis , Datos de Secuencia Molecular , Péptido Hidrolasas/metabolismo , Dodecil Sulfato de SodioRESUMEN
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in conjunction with enzymatic digestion of proteins and molecular weight search of peptide-mass database is a powerful technique for peptide/protein identification. Ideally, peptide mixtures should be compatible with both MALDI-TOF and microsequencing. In our laboratory, enzymatic digestion and extraction of peptides from polyvinylidene difluoride (PVDF)-bound proteins is performed in the presence of nonionic detergents. However, nonionic detergents have been shown to cause signal suppression in MALDI-TOF analysis. This study demonstrates that by using a modified matrix solution, peptide-mass fingerprinting of PVDF-bound proteins by MALDI-TOF can be obtained in the presence of nonionic detergents such as hydrogenated Triton X-100 (RTX-100), octylglucopyranoside, and Tween 20.