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Gene linkage of two‐dimensional polyacrylamide gel electrophoresis resolved proteins from isogene families in Saccharomyces cerevisiae by microsequencing of in‐gel trypsin generated peptides
Author(s) -
Norbeck Joakim,
Blomberg Anders
Publication year - 1995
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.1150160124
Subject(s) - saccharomyces cerevisiae , pulsed field gel electrophoresis , trypsin , gel electrophoresis , polyacrylamide gel electrophoresis , linkage (software) , gel electrophoresis of proteins , two dimensional gel electrophoresis , chemistry , biochemistry , gene , microbiology and biotechnology , biology , genetics , chromatography , enzyme , proteomics , genotype
The total cellular extract of proteins from the yeast Saccharomyces cerevisiae was resolved by preparative two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) where 500 μg was loaded per gel, and a number of proteins in isogene families were selected for microsequencing analysis. Peptides were generated from resolved proteins by in‐gel trypsin digestion, and fractionated by reversed phase‐high performance liquid chromatography (RP‐HPLC). Subsequent sequencing of peptides yielded internal amino acid sequences which unambiguously identified the selected proteins spots as gene products from PDC1, ENO1 ENO2, ADH1, HXK2, TDH2, TDH3, SSB1 and SSB2 . The chromatograms obtained from RP‐HPLC of related proteins were utilized to distinguish discriminating peptide fractions. With this approach two out of four amino acid differences between Ssb1p and Ssb2p were allocated. We estimate that by pooling five preparative gels, at least one hundred protein spots in the 2‐D pattern of S. cerevisiae will be obtained in sequencable amounts.