Amino acid analysis analysis

• Protein hydrolysis
• Identification of amino acid
• Chemical protein sequencing
  • Fast Atom Bombardment Mass Spectrometry
  • Sequencing strategy
  • Sequencing from gels

Protein hydrolysis

In order to determine the amino acid content it is necessary to break the protein chain down into its constituent amino acids by hydrolysis. The most common conditions for this are treatment with 6N HCl at 110^oC for 24 to 72 hours. The optimum time of hydrolysis has to be determined for each protein as the peptide bonds hydrolyse at different rates. Most are broadly similar but those between bulky hydrophobic residues take longer to hydrolyse.
Certain amino acids can be totally destroyed by hydrolysis if not protected. Tryptophan and tyrosine need to be protected from chlorine by use of thiol reagents or phenols as scavengers. Cysteine becomes oxidised on hydrolysis and it is necessary to pre-oxidise to cysteic acid before hydrolysis. A particular problem is that of deamination of amides - these are deamidated to the corresponding acidic residues. In this case determination of the amount of liberated ammonia is needed to quantify the amines.

Identification of amino acids

The amino acids liberated by hydrolysis are identified and quantified using chromatographic methods. Traditional methods of analysis involve ion-exchange chromatography and ninhydrin detection, automated in the amino acid analyser. Current methods can detect down to 50-100 pmol using ninhydrin and even lower using fluorescamine.
Modern chromatographic approaches are based upon HPLC using hydrophobic "reverse phase" columns. Several derivatisation chemistries are in common use: such as dansyl derivatives,o-pthalaldehyde (OPA) derivatives, phenylisothiocyanate (PITC) derivatives, or 9-fluorenylmethyl chloroformate (Fmoc) derivatives. The Fmoc procedure is one of the most widely used. Typical chromatogram of an amino acid analysis	Fmoc derivation of Amino Acids
Not all amino acids can be detected with the same sensitivity, different derivatisation chemistries giving differential sensitivity. For example, OPA-lysine derivatives are unstable and OPA can not detect proline or hydroxyproline unless they are previously oxidised with sodium hypochlorite. PITC derivatisation is not very good at detecting cysteines as cysteic acid and other forms of cysteine resulting from hydrolysis give poor separation on HPLC of the PTC-derivatives.

Chemical protein sequencing

Nowadays it is very common to sequence a protein by the DNA sequence encoding the protein. This, however, only possible if a cloned gene is available. It is often the case that chemical protein sequencing must be carried out in order to generate the oligonucleotide probes needed to clone the protein. Chemical sequencing can proceed via either the N-terminal or the C-terminal of the protein. N-terminal sequencing is much more common and is usually carried out by the Edman procedure.

Fast Atom Bombardment Mass Spectrometry

Fast Atom Bombardment Mass Spectrometry (FAB-MS) is is a very powerful technique utilising a stream of fast atoms such as argon to achieve ionisation in the mass spectrometer. This is a "soft" ionisation technique which generates sequence-specific fragment ions. The sequences of the fragments can be deduced from their masses. The technique is very sensitive and can handle mixtures of peptides. The only problem with this technique is its expense and the equipment is not always available.

A typical FAB-MS spectrum of a peptide

Sequencing strategy

Sequencing strategy using peptides

It is not feasible to sequence an entire protein molecule. The usual approach is to digest the protein with proteases to generate peptides and then sequence the peptides. The problem then is to order the peptides. This is achieved by the use of two peptides which cut the protein at different points, thus generating overlapping peptides. The peptides are separated by HPLC and sequenced. The sequence can be deduced from the overlapping peptides.

Sequencing from gels

A very convenient approach to the amino acid analysis and sequencing of proteins is the use of proteins separated on a PAGE or IEF gel. In order to perform the analyses, however, the proteins in the gel must be transfered to a polyvinylidene difluoride (PVDF) membrane. This is accomplished by electroblotting.

The protein bands can then be visualised using special stains such as Amido Black, Coomasie Blue R-250, Colloidal Gold or Ponceau S. proteins on the blots can then be hydrolysed by vapour phase HCL treatment and analysed for their amino acid content by Fmoc derivatives, or they can be sequenced using automated Edman sequencing directly from the band on the PVDF membrane. Such sequencing is usually only performed for about 15 cycles or so to generate a "sequence tag". This can then be used to generate an oligonucleotide probe for cloning or can be used to identify the protein via sequence analysis.

Electro-blotting