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 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.
Erdjument-Bromage
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Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Lui
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Sabatini
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Snyder
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Tempst
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D000595:Amino Acid Sequence; D001331:Automation; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D018797:Cell Cycle Proteins; D002851:Chromatography, High Pressure Liquid; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics; D011090:Polyenes / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017421:Sequence Analysis / Q000295:instrumentation / Q000379:methods; D020123:Sirolimus; D013056:Spectrophotometry, Ultraviolet; D022021:Tacrolimus Binding Proteins
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D006360:Heat-Shock Proteins; D007202:Indicators and Reagents; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011090:Polyenes; D029701:Saccharomyces cerevisiae Proteins; D020123:Sirolimus; D022021:Tacrolimus Binding Proteins; C083324:TOR1 protein, S cerevisiae; C081135:TOR2 protein, S cerevisiae