Peptide map analysis of recombinant human granulocyte colony stimulating factor: elimination of methionine...

Procedures for HPLC peptide map analysis of recombinant human granulocyte colony stimulating factor include reduction and S-carboxymethylation of the denatured protein, as well as protease digestion with Staphylococcus aureus endoproteinase Glu-C followed by reverse-phase liquid chromatographic separations. Under nonoptimized experimental conditions analytical problems including methionine modification during carboxymethylation, as well as generation of large, insoluble fragments and nonspecific cleavages during proteolytic digestion, occurred. These problems have complicated the analysis of peptide digests and affected the performance of HPLC columns. This report describes the elimination of these problems by optimizing peptide mapping procedures. We found that mild reduction and alkylation conditions can prevent methionine modification, while protease digestion in the presence of urea at room temperature alleviates generation of peptides derived from incomplete digestion and nonspecific cleavage by endoproteinase Glu-C. Peptide maps generated using the optimized procedures contain fewer peptide peaks with higher recovery. Elimination of incomplete digestion, which generates fewer larger, insoluble peptides, substantially extends the life of reverse-phase columns. The optimized method reproducibly produced peptide maps suitable for routine analysis.