HIGH-SENSITIVITY SEQUENCING OF LARGE PROTEINS - PARTIAL STRUCTURE OF THE RAPAMYCIN-FKBP12 TARGET

We report on studies leading to refinements of various steps of the pr otein internal sequencing process. Specifically, the developments comp rise (1) higher-sensitivity chemical sequencing through background red uction; (2) improved peptide recovery from rapid in situ digests of na nogram amount, nitrocellulose-bound proteins; and (3) accurate UV spec troscopic identification of Trp- and Cys-containing peptides. In addit ion, we describe strategies for 2-dimensional liquid chromatographic p eptide isolation from complex mixtures and a multi-analytical approach to peptide sequence analysis (Edman sequencing, matrix-assisted laser desorption mass spectrometry, and UV spectroscopy). Both strategies w ere applied in tandem to the primary structural analysis of a gel-puri fied, 250-kDa protein (mammalian target of rapamycin-FKBP 12 complex), available in low picomolar quantities only. More than 300-amino acids worth of sequence was obtained in mostly uninterrupted stretches, sev eral containing Trp, Cys, His, and Ser. That information has allowed t he matching of a biological function of a mammalian protein to a yeast gene product with a well-characterized mutant phenotype. The results also demonstrate that extended chemical sequencing analysis (e.g., 26 successive amino acids) is now feasible, starting with initial yields well below 1 pmol.