Two-dimensional gel electrophoresis of Caenorhabditis elegans homogenates and identification of protein spots by microsequencing.
Employing isoelectric focusing on immobilized pH gradients followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we have obtained a map of C. elegans proteins, from a mixed culture containing all developmental stages, presenting over 2000 spots within the window of isoelectric points (pI) 3.5-9 and a molecular mass of 10-200 kDa. Edman microsequencing yielded successful results in 12 out of 24 analyzed spots. All but one of the N-terminal sequences retrieved C. elegans sequences in cosmid and/or expressed sequence tag clones. Structurally related protein sequences found in data banks included enzymes in energy metabolism (cytochrome oxydase, ATP synthase, enolase), a fatty acid-binding protein, a translationally controlled tumor protein, an unknown C. elegans protein, an acidic ribosomal protein, a titin-like protein, a G-protein beta chain, cyclophilin, and cathepsin D. Experimental determination of N-termini allowed us to define sites of signal cleavage providing further information on the physiological role of the newly found C. elegans proteins. This report demonstrates the possibility of two-dimensional gel electrophoresis and Edman microsequencing in the elucidation of C. elegans proteome.
Bini
L
L
Department of Molecular Biology, University of Siena, Italy.
Heid
H
H
Liberatori
S
S
Geier
G
G
Pallini
V
V
Zwilling
R
R
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D015801:Helminth Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D017421:Sequence Analysis
D015801:Helminth Proteins