The purpose of this project is to develop the analytical technology required for the elution and subsequent microsequencing of proteins from two-dimensional polyacrylamide gels. A number of """"""""interesting"""""""" protein spots have been defined whose regulation is markedly altered during the multistep process of neoplastic transformation. Initially, microscale procedures aimed at the recovery and sequence analysis of these proteins from one- dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were investigated. Electroelution and passive extraction techniques were found to be suitable only when large amounts (> 500 pmoles) of protein were available. Electroblotting techniques in which the proteins are transferred to quaternary ammonium-derivatized glass fiber paper were modified such that 50 pmoles of soybean trypsin inhibitor applied to a ID- SDS-polyacrylamide gel could subsequently be correctly sequenced to 17 cycles. Other supports for protein electroblotting and subsequent microsequence analysis were investigated and Immobilon- P was found to be the support of choice. Using this support electrophoretic conditions were optimized to allow the N-terminal sequence analysis of up to 20 residues from 50-100 pmoles of protein applied to a 2D-gel. With ID-PAGE it was possible to obtain some sequence information from as little as 5 pmoles protein. This procedure was successfully applied to the sequence analysis of a number of previously uncharacterized membrane glyco- proteins. Work is currently underway to apply this technique to the analysis of other unknown proteins whose expression is altered during carcinogenesis and also to improve the sequencing yields of proteins isolated from two-dimensional polyacrylamide gels.
