Amino Acid,Proteins and Peptide Sequencing

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PRESENTED BY

SUPRIYA KUMARI

MSc Ist Yr

ROLL NO -410LS2055

Department of life science

NATIONAL INSTITUTE OF TECHNOLOGY

GUIDED BY- Dr. SAMIR PATRA SIR

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There are around 20 amino acids and all are amino acids .These are arranged in different sequences in different proteins so they exhibit different functions.

Sequencing of proteins are important tool for understanding cellular processes and allows drugs that target specific metabolic pathways to be invented more easily .It also determines which conformation the protein adopts and extent to which it is complexed with any non peptide molecule.

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FREDER ICK SANG ER first worked out the

sequence of amino acid residues in the polypeptide

chains of the hormone insulin.

The amino acid sequences of thousands of different

proteins from many species have been determined

using principles developed by Sanger .

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1. If the protein contains more than one polypeptide chain,

the chains are separated and purified.

2. Intrachain S-S (disulfide) cross-bridges between cysteine residues in the polypeptide chain are cleaved (If these

disulfides are interchain linkages,then step 2 precedes step 1.)

3. The amino acid composition of each polypeptide chain is

determined.

4. The N-terminal and C-terminal residues are identified.11/26/2010 4



5 . E ach polypeptide chain is cleaved into smaller fragments,

and the amino acid composition and sequence of

each fragment are determined.

6. Step 5 is repeated, using a different cleavage procedure

to generate a different and therefore overlapping set

of peptide fragments

7. The overall amino acid sequence of the protein is

reconstructed from the sequences in overlapping fragments.

8. The positions of S-S cross-bridges formed between

cysteine residues are located. 11/26/2010 5



The hormone insulin consists

of two polypeptide chains, A and B, held

together by two disulfide cross-bridges (S-S).

The A chain has 21 amino acid residues and an

intrachain disulfide; the B polypeptide contains

30

amino acids. The sequence shown is forbovine insulin.11/26/2010 7



A number of methods exist for cleaving

disulfides. An important consideration is to

carry out these cleavages so that the original or

even new S-S links do not form.

Oxidation of a disulfide by performic acid

results in the formation of two equivalents of

cysteic acid. Because these cysteic acid side

chains are ionized SO 3 _ groups, electrostatic

repulsion (as well as altered chemistry) prevents

S-S recombination. 11/26/2010 8



Oxidative cleavage by reaction with

performic acid.11/26/2010 9



The peptide is hydrolyzed into its constituent amino

acids by heating it in 6 N HCl at 110°C for 24 hours .

The complex amino acid mixture in the hydrolysate

obtained after digestion of a protein in 6 N HCl can be

separated into the component amino acids by either ion

exchange chromatography.

A comparision of chromatographic pattern with that of standard mixture of amino acid composition

of the peptide is done.

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E nd-group analysis reveals several things.F irst, it identifies the N- and C-terminal

residues in the polypeptide chain. Second,it can be a clue to the number of ends in the protein.

If the protein consists of two or more different polypeptide chains, then more than one end group may be discovered .

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F luorodinitrobenzene ( FD NB) was first used to

identify the N-terminal amino acid as it forms a

stable covalent bond with N-terminal residue of the

amino acid.D absyl chloride is now commonly used because it

forms fluorescent derivatives that can be detected with high sensitivity. It reacts with an uncharged -NH2

group to form a sulfonamide derivative that is stable

under conditions that hydrolyze peptide bonds.

Hydrolysis of our sample dabsyl-peptide in 6 N HCl would yield a dabsyl-amino acid, which could be

identified as dabsyl-alanine by its chromatographic

properties . 11/26/2010 12



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E dman degradation, has become the procedure of

choice. This method is preferable because it allows the

sequential identification of a series of residues

beginning at the N-terminus.In weakly basic solutions, phenylisothiocyanate, or

E dman·s reagent (phenyl-N=C=S), combines with the

free amino terminus of a protein, which can be excised from the end of the polypeptide chain and

recovered as a phenylthiohydantoin (PTH) derivative .

EDMAN DEGRADATION A chemical method devised by Pehr E dman is

usually employed .

This PTH derivative can be identified by

chromato ra hic methods 11/26/2010 15



N-Terminal analysis using Edman·s reagent, phenylisothiocyanate.Phenylisothiocyanate combines with the N-terminus of a peptide undermildly alkaline conditions to form a phenylthiocarbamoyl substitution. Upon

treatment with TFA (trifluoroacetic acid), this cyclizes to release the N-

terminal amino acid residue as a thiazolinone derivative, but the other

peptide bonds are not hydrolyzed. Organic extraction and treatment

with aqueous acid yield the N-terminal amino acid as a phenylthiohydantoinPTH derivative. 11/26/2010 16



C-TER MINAL ANALYSIS

F or the identification of the C-terminal residue of

polypeptides, an enzymatic approach is commonly used.

E NZYMATIC ANALYSIS WITH

CA R BOXYP E PTI D AS E S.

Carboxypeptidases are enzymes that cleave amino

acid residues from the C-termini of polypeptides in a

successive fashion.

F our carboxypeptidases are in general use: A, B,

C, and Y.

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carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues

Carboxypeptidase C (from citrus leaves) and carboxypeptidase from yeast act on any C-terminal

residue .

Carboxypeptidase A (from bovine pancreas) works

well in hydrolyzing the Cterminal peptide bond of all residues except proline, arginine, and lysine

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STEPS 5 AND 6. FRAGMENTATION OF THE

POLYPEPTIDE CHAIN

The cleavage methods employed are usually

enzymatic, but proteins can also be fragmented by

specific or nonspecific chemical means (such as partial

acid hydrolysis).

Proteolytic enzymes offer an advantage in that they

may hydrolyze only specific peptide bonds, and this specificity immediately gives information about the

peptide products

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A . TRYPSIN

Trypsin is specific in hydrolyzing only peptide bonds

in which the carbonyl function is contributed by an arginine or a lysine residue

Trypsin cleaves on the C-side of Arg or Lys,

generating a set of peptide fragments having Arg or Lys at their C-termini.

The number of smaller peptides resulting from trypsin action is equal to the total number of Arg

and Lys residues in the protein plus one³the

protein·s C-terminal peptide fragment 11/26/2010 20



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Chymotrypsin shows a strong preference for

hydrolyzing peptide bonds formed by the carboxyl groups of the aromatic amino acids, phenylalanine,

tyrosine,and tryptophan.

B . CHY MOTRYPSIN

Chymotrypsin produces a very different set of

products than trypsin, treatment of separate samples

of a protein with these two enzymes generates fragments whose sequences overlap

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C .CY A NOGEN B RO MIDE

CNBr acts upon methionine residues

The nucleophilic sulfur atom of Met reacts with

CNBr, yielding a sulfonium ion that undergoes a

rapid intramolecular rearrangement to form a cyclic iminolactone.

Water readily hydrolyzes this iminolactone, cleaving

the polypeptide and generating peptide fragments having C-terminal homoserine lactone residues at the

former Met positions .

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STEP 7. RECONSTRUCTION OF THE

OVERALL AMINO ACID SEQUENCE

The sequences obtained for the sets of fragments derived from two or more cleavage procedures are

now compared.

Peptides generated from specific hydrolysis of the polypeptide can be aligned to reveal the overall

amino acid sequence .

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Summary of the sequence analysis of catrocollastatin-C,

a 23.6-k D protein found in the venom of the western D iamondback rattlesnake Crotalus atrox.

Sequences shown are given in the one-letter amino acid code. The overall amino acid sequence (2 16 amino acid residues long) for catrocollastatin-C

as deduced from the overlapping sequences of peptide fragments is shown

on the lines headed CAT-C. The other Lines report the various sequences

used to obtain the overlaps.These sequences were obtained from (a) Nterm.:E dman degradation of the intact proteinin an automated E dman sequenator; (b) M:proteolytic fragments generated by CNBr

cleavage, followed by E dman sequencing of the individual

fragments (numbers denote fragments M 1 through M 5 );

(c) K: proteolytic fragments (K3 through K6) from endopeptidase Lys-C cleavage, followed by E dman sequencing;

(d) E : proteolytic fragments from Staphylococcus

protease ( E 13 through E 15 ) digestion of catrocollastatin sequenced in the

E dman sequenator . 11/26/2010 27



STEP 8. LOCATION OF DISULFIDE

CROSS-BRIDGES

If the primary structure includes disulfide bonds,their locations are determined in an additional step

after sequencing is completed

A sample of the protein is again cleaved with a reagent such as trypsin, this time without first

breaking the disulfide bonds .

The resulting peptides are separated by electrophoresis and compared with the original set

of peptides generated by trypsin

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F or each disulfide bond, two of the original peptides

will be missing and a new , larger peptide will appear.

The two missing peptides represent the regions of

the intact polypeptide that are linked by the

disulfide bond

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REFERENCES

GA RRE TT AN D G R ISHAM

BIOCH E MIST R Y

BY J ERE MY M B ER G,LUB ER T ST R Y ER

BIOCH E MIST R Y

BY L E HNING ER

WIKIP ED IA

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Exam time has come ,take care.

protect yourself from cold and flu11/26/2010 32

Amino Acid,Proteins and Peptide Sequencing

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