Identification of mouse liver proteins on two-dimensional electrophoresis gels by matrix-assisted laser desorption/ionization mass spectrometry of in situ enzymatic digests.
A number of proteins from a silver-stained two-dimensional (2-D) electrophoresis gel of mouse liver whole-cell lysate were identified by peptide mass mapping and sequence database searching. The excised protein spots were processed by in situ reduction and alkylation, followed by Lys-C digestion. The masses of the resulting peptide mixtures were measured with a matrix-assisted laser desorption/ionization (MALDI) reflection-time-of-flight mass spectrometer. These masses were used successfully to search a protein sequence database. Optimized silver staining and digestion protocols allowed proteins to be identified routinely at the low picomole level. The high mass accuracy and resolution provided by delayed extraction were important for high specificity in the database search. Fragment ion data obtained by MALDI post-source decay (PSD) measurements not only provided confirmation of peptide identification, but could be used to identify the protein from a single peptide without spectral interpretation.
O'Connell
K L
KL
Protein Chemistry Department, Genentech, Inc., South San Francisco, CA 94080, USA.
Stults
J T
JT
D000180:Acrylic Resins; D000595:Amino Acid Sequence; D000818:Animals; D016208:Databases, Factual; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D008099:Liver / Q000737:chemistry*; D008666:Metalloendopeptidases / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011506:Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000180:Acrylic Resins; D011506:Proteins; C016680:polyacrylamide gels; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase