COLLOIDAL SILVER STAINING OF ELECTROBLOTTED PROTEINS FOR HIGH-SENSITIVITY PEPTIDE-MAPPING BY LIQUID-CHROMATOGRAPHY ELECTROSPRAY-IONIZATION TANDEM MASS-SPECTROMETRY

Mass spectrometric techniques for the identification of proteins eithe r by amino acid sequencing or by correlation of mass spectral data wit h sequence databases are becoming increasingly sensitive and are rapid ly approaching the limit of detection achieved by the staining of prot eins in gels or, after electroblotting, on membranes. Here we present a technique for the sensitive staining of proteins electroblotted onto nitrocellulose or polyvinylidene difluoride membranes and enzymatic c leavage conditions for such proteins to achieve optimal recovery of pe ptides. The technique is based on the deposition of colloidal silver o n the membrane-bound proteins. Peptide mixtures generated by proteolys is on the membrane were recovered at high yields and were compatible w ith analysis by reverse-phase chromatography and on-line electrospray ionization mass spectrometry. This simple and rapid colloidal silver s taining procedure allowed the visualization of less than 5 ng of prote in in a band and thus approached the sensitivity of silver staining in gels. We demonstrate that this method allows the detection of subpico mole amounts of electroblotted proteins and their identification by hi gh-performance liquid chromatography-electrospray ionization tandem ma ss spectrometry. (C) 1997 Academic Press.