Post on 12-Apr-2017
Protein SequencingBy
Mohammed z. Naji
Schematic amino acid R groups
A AlaC CysD AspE GluF Phe*G GlyH His*I Ile*K Lys*L Leu*M Met*N AsnP ProQ GlnR Arg*S SerT Thr*V Val*W Trp*Y Tyr
C N O S
All amino acids absorb in infrared
region Only Phe, Tyr, and Trp absorb UV Absorbance at 280 nm is a good
diagnostic device for amino acids
Amino acid detection
Aromatics absorb UV
Ninhydrin
is a chemical used to detect ammonia or primary and secondary amines.
Mikhail Tswett father of ‘chromatography’ Chromatography
Ion exchange chromatography (net ionic character) High-performance liquid chromatography (HPLC) Gas chromatography
Electrophoresis(charge/size) Paper (ninhydrin) Capillary electrophoresis (fluorescence, UV, laser-
induced capillary vibration)
Separation of amino acids
Chromatography
Detection: refractive index, circular dichroism, (MS/MS) vs. derivitization for UV or fluorescence or MS
Chromatography
Elution order depends on affinity to the matrix!!
Proteins
(polypeptides)
100 amino acid protein has 20100
combinations 1953 Frederick Sanger sequenced the
two chains of insulin (21 aa) All of the molecules of a given protein
have the same sequence Proteins can be sequenced in two ways:
- direct amino acid sequencing- indirect sequencing of the encoding
gene (DNA)
Sequencing
Sequences and composition often (not always)
reflect the function of the protein (often proteins of similar function will have similar sequences)
Homologous proteins from different organisms have homologous sequences
Why does sequence matter?
Changes in protein sequence can be
used to infer evolutionary relationships
1. If more than one polypeptide chain,
separate.2. Cleave (reduce) disulfide bridges3. Determine composition of each
chain4. Determine N- and C-terminal
residues
Conventional Sequencing
5. Cleave each chain into smaller
fragments and determine the sequence of each chain
6. Repeat step 5, using a different cleavage procedure to generate a different set of fragments.
Conventional Sequencing
7. Reconstruct the sequence of the
protein from the sequences of overlapping fragments
8. Determine the positions of the disulfide crosslinks
Conventional Sequencing
Subunit interactions depend on weak forces
Separation is achieved with:- extreme pH- 8M urea- 6M guanidine HCl- high salt concentration (usually
ammonium sulfate)
Separation of chains
Performic acid oxidation Sulfhydryl reducing agents
- mercaptoethanol• alkylate (iodoacetate) prevents
recombination
Cleavage of Disulfide bonds
Enzymatic fragmentation
Trypsin (R or K) Chymotrypsin (F or Y or W)
Chemical fragmentation cyanogen bromide (Methomoserine lactone)
Fragmentation of the chains
Trypsin digestion
Enzymatic analysis (carboxypeptidase)
Carboxypeptidase A cleaves any residue except Pro, Arg, and Lys
Carboxypeptidase B (hog pancreas) only works on Arg and Lys
Carboxypeptidase C, Y any residue
C-terminal analysis
Mass spectrometry