1. The Covalent Structure of Proteins

2. 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

3. Determination of Amino Acid Sequence
Sanger
Edman

4. 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

5. Breaking Disulfide Bonds

6. Sequencing Large Proteins

7. 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

8. 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

9. 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

10. MS/MS

11. 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

12. Chemical Synthesis of Small Peptide

13. Chemical Synthesis of Small Peptide