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