Download Determination of Amino Acid Sequence

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