Structural analysis and identification of gel-purified proteins, available in the femtomole range, using a novel computer program for peptide sequence assignment, by matrix-assisted laser desorption ionization-reflectron time-of-flight-mass spectrometry.
A procedure is described for structural characterization and identification of proteins, purified by either one- or two-dimensional gel electrophoresis in the low picomole to femtomole range. The purified proteins are first detected in the primary gels by the sensitive reverse staining procedure described by Fernandez-Patron et al. (Anal. Biochem. 1995, 224, 203-211) and consecutively reeluted from combined get pieces and concentrated in the tip of a Pasteur pipette in a secondary gel matrix consisting of either sodium dodecyl sulfate-polyacrylamide or agarose. The concentrated proteins are in-matrix-digested and the resulting peptides are separated by reverse-phase high performance liquid chromatography (HPLC) combined with microsequencing or analyzed by matrix-assisted laser desorption ionization--time of flight--mass spectrometry. Protein identification is based on sequence homology or on the peptide mass pattern. The matching peptide sequences can additionally be verified by matching their measured post-source decay spectra with the calculated fragmentation patterns of the isobaric candidate peptides appearing on the search list. This is done by a computer program referred to as MassFrag, described in this paper. We demonstrate that it is possible to identify protein that are only available in the femtomole range and whose sequences are stored in nonredundant protein databases or nucleotide and expressed sequence tag databases.
Gevaert
K
K
Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Belgium.
Verschelde
J L
JL
Puype
M
M
Van Damme
J
J
Goethals
M
M
De Boeck
S
S
Vandekerckhove
J
J
D000595:Amino Acid Sequence; D003201:Computers; D001330:Electronic Data Processing; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005782:Gels; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D005782:Gels