The Covalent Structure of Proteins

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Presentation transcript:

The Covalent Structure of Proteins

Determination of Amino Acid Sequence Determination of amino acid sequence from a protein Sanger’s method N-terminal labeling and identification Using FDNB (1-fluoro-2,4-dinitrobenzene) Edman Degradation Sequencing of the entire polypeptide Sequential labeling and removal of the N-terminal amino acid Sequenator Automated sequencing of proteins Accuracy is depending on the efficiency of the individual chemical step > 99% in modern sequenator Translation from DNA sequence DNA sequence  protein sequence Protein sequence  cloning of the gene

Determination of Amino Acid Sequence Sanger Edman

Sequencing Large Proteins Breaking disulfide bonds Oxidation by performic acid Reduction and carboxymethylation Cleaving the polypeptide chain Using proteases Cleavage of peptide bond next to particular amino acid residues Trypsin: Lys, Arg Sequencing of peptides Ordering peptide fragments Compare sequences generated from different cleavage methods Locating disulfide bonds Comparison of cleavage fragment with or without breaking disulfide bonds

Breaking Disulfide Bonds

Sequencing Large Proteins

Determination of Amino Acid Sequences Using Mass spectroscopy Components of mass spectrometer Ionizer: converting molecules to gas phase ions Soft ionizer for large molecules MALDI MS ESI MS Mass analyzer: separate the ions according to the m/z Time of flight (TOF) Measuring the time take by ions to travel to the detector Ion detector Mass spectrometer for protein analysis Determination of molecular weight Determination of short polypeptide sequence Tandem MS, MS/MS

MALDI-TOF Matrix-assisted laser desorption/ionization mass spectrormetry Protein placed in a light-absorbing matrix Ionization and desorption of proteins by a short pulse of laser

ESI-TOF Electrospary ionization mass spectrometry Passing of analyte solution through a charged needle with a high electrical potential Dispersion of charged microdroplets John Fenn(2002) Virginia Commonwealth University

MS/MS

Chemical Synthesis of Small Peptide Developed by R. Bruce Merrifield (1962) Synthesis from C- to N- terminal on an polymer support Fmoc (9-fluorenylmethoxycarbonyl) Protection of unwanted reaction

Chemical Synthesis of Small Peptide

Chemical Synthesis of Small Peptide