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High-Sensitivity sequencing of large proteins: Partial structure of the rapamycin-fkbp12 target by Hediye Erdjument-Bromage; Mary Lui; Paul Tempst; David M. Sabatini; Solomon H. Snyder is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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## Abstract 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

Who reads High-Sensitivity sequencing of large proteins: Partial structure of the rapamycin-fkbp12 target?

Author

Hediye Erdjument-Bromage; Mary Lui; Paul Tempst; David M. Sabatini; Solomon H. Snyder

Publisher

Cold Spring Harbor Laboratory Press; Wiley (John Wiley & Sons); Wiley-Blackwell; Wiley (ISSN 0961-8368)

Published

1994

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)