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Lec10 - CNBr cleavage Phe-Trp-Met Gly-Ala-Lys-Leu-Pro-Met...

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Protein Sequencing: Overview
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Cleave disulfide bonds Cleave protein into smaller fragments using specific proteases or chemicals Purify the fragments Edman degradation of individual fragments or sequencing by mass-spectrometry Protein/peptide Sequencing
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Dansyl Chloride Reaction
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Edman Degradation
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Peptide Bond Cleavage of an Endopeptidase
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Proteases Endopeptidases a. Pepsin b. Trypsin c. Chymotrypsin d. Thermolysin e. Endopeptidase V8 f. LysC (Cleaves after a lysine) g. Elastase Exopeptidase a. Carboxypeptidase (Cleaves a peptide sequentially from the C-terminus)
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Various Endopeptidase
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Peptide Cleavage by CNBr
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Endgroup analysis Modify the N-terminal residues Hydrolyze all peptide bonds Identify the modified amino acids Does the protein contain one or more polypeptides?
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Amino Acid Analysis
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Amino Acid Sequencing by Overlapping Cleavage
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Cleavage of Disulfide Bonds
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CNBr cleavage: Phe-Trp-Met Gly-Ala-Lys-Leu-Pro-Met Asp-Gly-Arg-Cys-Ala-Gln Trypsin cleavage: Phe-Trp-Met-Gly-Ala-Lys Leu-Pro-Met-Asp-Gly-Arg Cys-Ala-Gln
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Unformatted text preview: CNBr cleavage: Phe-Trp-Met Gly-Ala-Lys-Leu-Pro-Met Asp-Gly-Arg-Cys-Ala-Gln Trypsin cleavage: Phe-Trp-Met-Gly-Ala-Lys Leu-Pro-Met-Asp-Gly-Arg Cys-Ala-Gln Gly-Ala-Lys-Leu-Pro-Met Leu-Pro-Met-Asp-Gly-Arg Phe-Trp-Met-Gly-Ala-Lys Asp-Gly-Arg-Cys-Ala-Gln Electrospray Ionization MS ESI-MS of apomyoglobin Tandem Mass Spectrometry Protein Sequence Database Protein evolution Random mutations result in changes in protein sequence Reduced fitness loss of that mutant Increased fitness increased frequency Proteins slowly change during evolution invariant residues conservatively substituted residues hypervariable residues Comparing sequences of the same protein in different species shows how a protein has changed Phylogenetic Tree Rates of Evolution Mechanisms of Evolution Gene duplication Domain swapping Point mutations Natural selection Genealogy of a gene family Modular Construction of Proteins...
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