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 β 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.