Peptide analysis

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6.1. AA Composition

 A protein or peptide is initially subjected to complete hydrolysis to determine it’s constituent AAs.

 Complete hydrolysis is performed in either strong acid or strong alkali.

 The relative amount of each AA may them be determined by chromatographic techniques.

 Acid Hydrolysis

o The peptide is heated with excess 6M HCl at 100oC for 10 to 24 hours.

o Excess HCl is removed and AAs remain as their HCl salts.

o The advantage of acid hydrolysis is that there is little or no racemization of AAs.

o The disadvantage is that, Trp is destroyed and Gln is converted to Glu and Asn to Asp.

 Alkaline Hydrolysis

o The advantage of boiling with alkali (NaOH) is that Trp remains stable.

o This technique is not commonly used since all AAs are racemized and Cys, Cystine, Ser, and Thr are destroyed.

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6.2. Sequencing of a Peptide

 Complete mixtures of peptides resulting from partial hydrolysis of proteins are much more difficult to separate than mixtures of AAs because the number of possible peptides is very much greater than the known number of AAs in proteins Separation and purification of peptides in a mixture by paper electrophoresis into neutral, acidic and basic at pH 6. At this pH:

i. Acidic peptides (those with an excess of Glu or Asp) will be negatively charged and move to the anode;

ii. Basic peptides (those with an excess Arg, Lys or His) will be positively charged and migrate to the cathode;

iii. Neutral peptides (those with no acidic or basic AA’s or an equal number of them) will not migrate to any extent.

 Following this gross separation of peptide types; individual peptides can be better separated by further electrophoresis at an appropriate pH; acid peptides at low pH; basic types at high pH and so on.

 Alternatively, the broad groups of peptides separated by initial electrophoresis at pH 6 may be separated further by ion exchange chromatography.

 Once all peptides in a mixture are separated. Individual peptides can be completely hydrolyzed and its amino acid composition determined as described previously.

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6.3. Incomplete hydrolysis of proteins to produce medium/small sized peptides.

 Where a polypeptide chain is to be studied, the entire sequence may comprise 100 or more AAs. In such cases, it is necessary to subdivide the sequence and to sequence each of the resulting fragments individually. Thereafter, subsequent steps allow determination of the entire sequence.

 This is achieved using enzymes known as endopeptidases, which hydrolyze specific internal peptide bonds in the central sections of a polypeptide chain (i.e. not at the ends or termini).

Table: Specificity of cleavage procedures utilized in Peptide Sequence Analysis

PROCEDURE

SITE

SPECIFICITY

TERMINAL CLEAVAGES

Edman Degradation

C – side of N terminus

Rn = any AA

Leucine Aminopeptidase

C – side of N terminus

Rn = any AA except Pro

Carboxypeptidase A

N – side of C terminus

Rn = any AA except Arg, Lys, Pro

Carboxypeptidase B

N – side of C terminus

Rn = Arg, Lys, and Pro

Hydrazinolysis

N – side of C terminus

Rn = any AA

INTERNAL CLEAVAGES

Cyanogen bromide

C – side of residue Rn

Rn = Met

Cyanogen bromide anhydrous

C – side of residue Rn

Rn = Met and Trp

Trypsin

C – side of residue Rn

Rn = Lys and Arg

Chymotrypsin

C – side of residue Rn

Rn = Phe, Trp, Tyr, and Leu

Staphylococcal Protease V8

C – side of residue Rn

Rn = Asp and Glu

Pepsin

N – side of residue Rn

Rn = Leu, Asp, Glu, Phe, Tyr, and Trp

Thermolysin

N – side of residue Rn

Rn = Leu, Ile, Val, Phe, Tyr, and Trp

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6.4. Identification of amino terminal AA residue of a Peptide

a) Chemical Analysis: i) Sanger reaction

ii) Dansyl Chloride reaction

iii) Edman reaction

N.B. – Only the N-terminal residue will react. Thereafter the peptide chain is hydrolyzed (6M HCl) completely. The individual AAs are analyzed by chromatography and the AA derivative resulting from any one of the reactions above is derived from the amino terminal residue (i.e. AA number 1).

b) Enzymatic analysis

- The exopeptidase leucine aminopeptidase is employed. It removes the N-terminal residue and leaves the rest of the polypeptide chain intact. The released amino acid may be analyzed and identified via chromatographic techniques.

- In the case of an N-terminal prolyl residue, the peptide link is not digested.

- N.B. A disadvantage exists in this method, in that the reaction is progressive. That is as soon as the first N-terminal residue is released another becomes available and is also cleaved. To an extent this limitation can be overcome by precise timing of residue release and immediate processing thereafter.

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The Edman Degradation Procedure

 This technique was developed by Pehr Edman in 1950 and it permits removal and identification of one residue at a time from the N-terminus of a polypeptide.

 The Edman Degradation Procedure involves treating a protein or polypeptide with PITC also known as the Edman reagent at pH9. The reagent may also be employed in the measurement of free AAs. PTC-AA derivatives can be quantified (picomole or 1012 mol) spectrophotometrically at 254nm.

 Determining the AA sequence of polypeptides

 PITC reacts with the free N-terminus of the chain to form Phenylthiocarbamoyl (PTC)-peptide.

 When the PTC-peptide is treated with an anhydrous acid (trifluoroacetic acid), the peptide bond of the N-terminal AA is selectively cleaved, releasing anilinothiazolinone derivative of the N-terminal residue.

 This derivative can be extracted with an organic solvent such as butyl chloride, leaving the remaining peptide in the aqueous phase.

 The above derivative is unstable and is treated with HCl which converts it to the stable 3-phenyl-2-hydantoin (PTH) derivative of the N-terminal AA, which can be identified chromatographically.

 The remaining polypeptide chain (one residue shorter) can be adjusted to pH 9 and treated again with PITC and the procedure repeated as for the initial AA. This entire procedure can be repeated serially using an automated instrument known as the sequenator.

 A few exceptional peptides exist which may not be sequenced with this technique.

o Cyclic peptides (gramicidin)

o Peptides in which the amino terminus as been modified e.g. by acetylation as in the Tobacco Mosaic Viral protein.

 The yield for the Edman degradation procedure under carefully controlled conditions approaches 100% and a few picomoles of sample protein can yield sequences for 30 residues or more before further measurement is obscured by the increasing concentration of unrecovered sample form previous cycles of the procedure. For example, if the Edman degradation procedure has an efficiency of 98%, the cumulative yield at the 30th cycle would be 0.983830 or 0.55. In other words, only about half of the PTH-AA’s generated in the 30th cycle would be derived from the 30 residue from the N-terminus.

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6.5. Identification of carboxyl terminal AA residue of a Peptide

a) Chemical Analysis

i) Reduction with LiBH4 or NaBH4

ii) Hydrazinolysis

N.B. – Only the C-terminal residue will react. Thereafter the peptide chain is hydrolyzed (6M HCl) completely. The individual AAs are analysed by chromatography and the AA derivative resulting from any one of the reactions above is derived from the carboxyl terminal residue.

b) Enzymatic analysis

- The exopeptidase Carboxypeptidase A, B, or C is employed. It removes the C-terminal residue and leaves the rest of the polypeptide chain intact. The released amino acid may be analyzed and identified via chromatographic techniques.

- In the case of C-terminal modification as in Oxytocin or Vasopressin the carboxyl terminal peptide link is not digested.

- N.B. A disadvantage exists in this method, in that the reaction is progressive. That is as soon as the first C-terminal residue is released another becomes available and is also cleaved. To an extent this limitation can be overcome by precise timing of residue release and immediate processing therafter.

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6.6. Peptide sequence determination

 The following steps are applicable to the sequence determination of a large polypeptide or protein consisting of a single strand of AAs. All techniques mentioned are explained in preceding sections.

i) The amino and carboxyl termini of the polypeptide are identified.

ii) The intact peptide is cleaved into a series of smaller peptides, using trypsin.

iii) Peptide fragments from (ii) are separated out by electrophoresis or other methods.

iv) The amino and carboxyl termini of each fragment from (ii) are identified.

v) Samples of each fragment from (ii) are hydrolyzed completely and their compositions determined.

 From the data obtained thus far, the AA sequence of any di- and tripeptide fragments will be known; for larger fragments the amino and carboxyl termini will be determined and AA composition of the entire polypeptide will be understood.

vi) The peptide fragments (up to 30 AAs) from (ii) are now subjected to Edman degradation and their sequences determined.

vii) Employing chymotrypsin or pepsin, another intact sample of the original polypeptide is at this stage cleaved into a different series of fragments.

viii) This second set of fragments is separated and analysed exactly as for the first set i.e. steps (iii), (iv), (v), and (vi) is repeated.

ix) By comparing the AA sequences of the two sets of fragments, taking particular note of overlapping sequences and common termini, the peptide fragments can usually be ordered into the correct sequence, thereby generating the sequence of AAs for the entire polypeptide.