William E. Werner

A strategy of obtain internal sequence information from blotted proteins after initial N-terminal sequencing

Publisher Summary To generate internal peptide fragments for the identification of sequences from N-terminally blocked proteins, or for the maximization of sequence, information from larger proteins require purification of additional protein sample. With the advent of high sensitivity sample preparation systems employing capillary HPLC, it has become feasible to explore the generation and purification of internal peptide fragments from modest amounts of protein (60 picomole) immobilized onto polyvinylidene difluoride (PVDF) membrane that have previously been subjected to Edman degradation. Initial investigations reveal that after proteins have subjected to Edman chemistry, they are refractory to digestion by the enzymes trypsin, Lys-C, and Glu-C. It is possible to generate internal fragments using chymotrypsin, but the extensive auto-digestion products contaminate the subsequent peptide maps. Chemical cleavage methods were employed resulting in great success. Two proteins—that is, carbonic anhydrase and transferrin, are chosen as models for this study. The experiments discussed in this chapter demonstrates the generation, extraction, and the subsequent purification strategy of internal fragments using both cyanogen bromide to cleave proteins at methionine, and incubation in formic acid at elevated temperature to cut between the aspartic acid and proline. There are two advantages to performing cyanogen bromide digestions in 70% formic acid at an elevated temperature: first, the methionine specific cleavage occurred faster, and second, the cleavage between aspartic acid and proline pairs were catalyzed. This resulted in the generation of more peptide fragments for all samples tested in a relatively short time.