We report on studies leading to refinements of various steps of the protein internal sequencing process. Specifically, the developments comprise (1) higher‐sensitivity chemical sequencing through background reduction; (2) improved peptide recovery from rapid in situ digests of nanogram amount, nitrocellulose‐bound proteins; and (3) accurate UV spectroscopic identification of Trp‐ and Cys‐containing peptides. In addition, we describe strategies for 2‐dimensional liquid chromatographic peptide 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 were applied in tandem to the primary structural analysis of a gel‐purified, 250‐kDa protein (mammalian target of rapamycin‐FKBP12 complex), available in low picomolar quantities only. More than 300‐amino acids worth of sequence was obtained in mostly uninterrupted stretches, several containing Trp, Cys, His, and Ser. That information has allowed the 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.
- 250‐kDa protein
- chemical sequencing; in situ proteolysis
- liquid chromatography
- matrix‐assisted laser‐desorption mass spectrometry
- ultraviolet spectroscopy
ASJC Scopus subject areas
- Molecular Biology