Abstract
Cross-linking mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid compositions of some protein regions impede the detection of cross-linked residues, although it would yield invaluable information for protein modeling. Here, we report on a sequential-digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin-cleavage sites. We exploited intrinsic substrate-recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Chromatography, Liquid
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Cross-Linking Reagents / chemistry
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Humans
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Pancreatic Elastase / chemistry*
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Peptides / analysis
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Peptides / chemistry
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Proteolysis
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Sf9 Cells
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Spodoptera
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Succinimides / chemistry
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TATA-Binding Protein Associated Factors / analysis*
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TATA-Binding Protein Associated Factors / chemistry
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Tandem Mass Spectrometry / methods
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Transcription Factor TFIID / analysis*
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Transcription Factor TFIID / chemistry
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Trypsin / chemistry*
Substances
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Cross-Linking Reagents
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Peptides
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Succinimides
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TAF4 protein, human
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TATA-Binding Protein Associated Factors
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Transcription Factor TFIID
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bis(sulfosuccinimidyl)suberate
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Pancreatic Elastase
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Trypsin