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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‐fligh — mass spectrometry
Author(s) -
Gevaert Kris,
Verschelde JeanLuc,
Puype Magda,
Van Damme Jozef,
Goethals Marc,
de Boeck Stefaan,
Vandekerckhove Joël
Publication year - 1996
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150170513
Subject(s) - chromatography , chemistry , mass spectrometry , isobaric labeling , matrix assisted laser desorption/ionization , peptide mass fingerprinting , peptide , peptide sequence , gel electrophoresis , protein mass spectrometry , analytical chemistry (journal) , electrospray ionization , biochemistry , desorption , proteomics , organic chemistry , adsorption , gene
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 gel pieces and concentrated in the tip of a Pasteur pipette in a secondary gel matrix consisting of either sodium dodecyl sulfatepolyacrylamide 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.