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 either by amino acid sequencing or by correlation of mass spectral data with sequence databases are becoming increasingly sensitive and are rapidly approaching the limit of detection achieved by the staining of proteins 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 cleavage conditions for such proteins to achieve optimal recovery of peptides. The technique is based on the deposition of colloidal silver on the membrane-bound proteins. Peptide mixtures generated by proteolysis on the membrane were recovered at high yields and were compatible with analysis by reverse-phase chromatography and on-line electrospray ionization mass spectrometry. This simple and rapid colloidal silver staining procedure allowed the visualization of less than 5 ng of protein in a band and thus approached the sensitivity of silver staining in gels. We demonstrate that this method allows the detection of subpicomole amounts of electroblotted proteins and their identification by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.
van Oostveen
I
I
Department of Molecular Biotechnology, University of Washington, Seattle 98195, USA.
Ducret
A
A
Aebersold
R
R
D000580:Amido Black; D000595:Amino Acid Sequence; D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D003102:Colloids; D013058:Mass Spectrometry / Q000379:methods; D008832:Microchemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods* / Q000706:statistics & numerical data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012834:Silver; D013194:Staining and Labeling / Q000379:methods* / Q000706:statistics & numerical data; D014357:Trypsin
D003102:Colloids; D010455:Peptides; D011506:Proteins; D012834:Silver; D014357:Trypsin; D002256:Carbonic Anhydrases; D000580:Amido Black