Protein sequencing

Protein sequencing is a technique to determine the amino acid sequence of a protein, as well as which conformation the protein adopts and the extent to which it is complexed with any non-peptide molecules

 The two major direct methods of protein sequencing are mass spectrometry and the Edman degradation reaction It is also possible to generate an amino acid sequence from the DNA or mRNA sequence encoding the protein

Determining amino acid composition

Desirable to know the unordered amino acid composition of a protein prior to attempting to find the ordered sequence, as this knowledge can be used to facilitate the discovery of errors in the sequencing process or to distinguish between ambiguous results Knowledge of the frequency of certain amino acids may also be used to choose which protease to use for digestion of the protein.

 Amino acid analysis method for determining amino acid frequency is as follows: Hydrolyse a known quantity of protein into its constituent amino acids. Separate the amino acids

Hydrolysis
Hydrolysis is done by heating a sample of the protein in 6 Molar hydrochloric acid to 100110 degrees Celsius for 24 hours or longer Some amino acids (serine, threonine, tyrosine, tryptophan, glutamine and cystine) are degraded

Separation
Amino acids can be separated by ion-exchange chromatography or hydrophobic interaction chromatography Ion-exchange chromatography : sulfonated polystyrene as a matrix, adding the amino acids in acid solution and passing a buffer of steadily increasing pH through the column. Amino acids will be eluted when the pH reaches their respective isoelectric points Hydrophobic interaction chromatography : reversed phase chromatography using C8 and C18 silica columns separate amino acids in solution in less than 40 minutes through the use of an optimised elution gradient

Quantitative analysis
Respective quantities are determined by adding a reagent that will form a coloured derivative Eg. Ninhydrin - gives a yellow colour when reacted with proline, and a vivid purple with other amino acids

N-terminal amino acid analysis

Aids the ordering of individual peptide fragments' sequences into a whole chain First round of Edman degradation is often contaminated by impurities and therefore does not give an accurate determination of the Nterminal amino acid

 A generalised method for Nterminal amino acid analysis

React the peptide with a reagent which will selectively label the terminal amino acid. Hydrolyse the protein. Determine the amino acid by chromatography and comparison with standards

Different reagents can be used to label terminal amino acids

Sanger's reagent (1-fluoro2,4-dinitrobenzene) Dansyl chloride Phenylisothiocyanate

C-terminal amino acid analysis

Add carboxypeptidases to a solution of the protein Take samples at regular intervals Determine the terminal amino acid by analysing a plot of amino acid concentrations against time

Edman degradation
allows the ordered amino acid composition of a protein to be discovered Automated Edman sequencers sequence peptides up to approximately 50 amino acids long

Steps in Edman degradation

Break any disulfide bridges in the protein with an oxidising agent like performic acid or reducing agent like 2-mercaptoethanol. A protecting group such as iodoacetic acid may be necessary to prevent the bonds from re-forming. Separate and purify the individual chains of the protein complex, if there are more than one. Determine the amino acid composition of each chain. Determine the terminal amino acids of each chain. Break each chain into fragments under 50 amino acids long. Separate and purify the fragments. Determine the sequence of each fragment. Repeat with a different pattern of cleavage. Construct the sequence of the overall protein. Digestion is done either by endopeptidases such as trypsin or pepsin or by chemical reagents such as cyanogen bromide. Different enzymes give different cleavage patterns, and the overlap between fragments can be used to construct an overall sequence

Edman degradation reaction

Peptide to be sequenced is adsorbed onto a solid surface one common substrate is glass fibre coated with polybrene, a cationic polymer Edman reagent, phenylisothiocyanate (PITC), is added to the adsorbed peptide, together with a mildly basic buffer solution of 12% trimethylamine. This reacts with the amine group of the N-terminal amino acid The terminal amino acid can then be selectively detached by the addition of anhydrous acid The derivative then isomerises to give a substituted phenylthiohydantoin which can be washed off and identified by chromatography, and the cycle can be repeated

Mass spectrometry
Peptides are also easier to prepare for mass spectrometry than whole proteins, because they are more soluble Method of delivering the peptides to the spectrometer is electrospray ionization The protein is digested by an endoprotease, and the resulting solution is passed through a high pressure liquid chromatography column At the end of this column, the solution is sprayed out of a narrow nozzle charged to a high positive potential into the mass spectrometer

 The charge on the droplets causes them to fragment until only single ions remain The peptides are then fragmented and the mass-tocharge ratios of the fragments measured The mass spectrum is analysed by computer and often compared against a database of previously sequenced proteins in order to determine the sequences of the fragments This process is then repeated with a different digestion enzyme, and the overlaps in the sequences are used to construct a sequence for the protein

Predicting protein sequence from DNA/RNA sequences