High-sensitivity sequencing of large proteins: Partial structure of the rapamycin-FKBP12 target

H. Erdjument-Bromage, M. Lui, D. M. Sabatini, S. H. Snyder, Solomon H Snyder

Research output: Contribution to journalArticle

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 also demonstrate that extended chemical sequencing analysis (e.g., 26 successive amino acids) is now feasible, starting with initial yields well below 1 pmol.

Original languageEnglish (US)
Pages (from-to)2435-2446
Number of pages12
JournalProtein Science
Volume3
Issue number12
StatePublished - 1994

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Tacrolimus Binding Protein 1A
Protein Sequence Analysis
Sirolimus
Peptides
TOR Serine-Threonine Kinases
Proteins
Collodion
Complex Mixtures
Amino Acids
Amino Acid Sequence
Mass Spectrometry
Spectrum Analysis
Lasers
Yeasts
Ultraviolet spectroscopy
Gels
Structural analysis
Yeast
Mass spectrometry
Phenotype

ASJC Scopus subject areas

  • Biochemistry

Cite this

Erdjument-Bromage, H., Lui, M., Sabatini, D. M., Snyder, S. H., & Snyder, S. H. (1994). High-sensitivity sequencing of large proteins: Partial structure of the rapamycin-FKBP12 target. Protein Science, 3(12), 2435-2446.

High-sensitivity sequencing of large proteins : Partial structure of the rapamycin-FKBP12 target. / Erdjument-Bromage, H.; Lui, M.; Sabatini, D. M.; Snyder, S. H.; Snyder, Solomon H.

In: Protein Science, Vol. 3, No. 12, 1994, p. 2435-2446.

Research output: Contribution to journalArticle

Erdjument-Bromage, H, Lui, M, Sabatini, DM, Snyder, SH & Snyder, SH 1994, 'High-sensitivity sequencing of large proteins: Partial structure of the rapamycin-FKBP12 target', Protein Science, vol. 3, no. 12, pp. 2435-2446.
Erdjument-Bromage, H. ; Lui, M. ; Sabatini, D. M. ; Snyder, S. H. ; Snyder, Solomon H. / High-sensitivity sequencing of large proteins : Partial structure of the rapamycin-FKBP12 target. In: Protein Science. 1994 ; Vol. 3, No. 12. pp. 2435-2446.
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