AMINO ACID SEQUENCING

MADE BY:MADE BY:VAISHALI JAINVAISHALI JAINROLL NO: 06ROLL NO: 06

DEFINITIONPeptide sequence, or amino acid sequence, is the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides and proteins. The sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group.Shorter peptides are sequenced with automated procedures but larger proteins must be sequenced in smaller segments.

IMPORTANCE OF AMINO ACID SEQUENCING

• Knowledge of a protein’s amino acid sequence is prerequisite for determining its three-dimensional structure and is essential for understanding its molecular mechanism of action.• Many inherited diseases are caused by mutations that result in an

amino acid change in a protein. Amino acid sequence analysis can assist in the development of diagnostic tests and effective therapies.• Sequence comparisons among analogous proteins from different

species yield insights into protein function and reveal evolutionary relationships among the proteins and the organisms that produce them.

HISTORY Frederick Sanger determined the first known protein sequence, that of hormone insulin in 1953. Since then, many additional proteins have been sequenced, and the sequences of many more proteins have been inferred from their DNA sequences.

N-TERMINAL ANALYSISEach polypeptide chain (if it is not chemically blocked) has an N-terminal residue. Identifying this “end group” can establish the number of chemically distinct polypeptides in a protein. For example, insulin has equal amounts of the N-terminal residues Gly and Phe, which indicates that it has equal numbers of two nonidentical polypeptide chains.

METHODS TO DETERMINE N-TERMINAL AMINO ACID

• SANGER’S METHOD: Sanger developed the reagent 1-fluoro-2,4-dinitrobenzene (FDNB). After treatment of protein with FDNB , the amino terminal residue is labeled with FDNB and the polypeptide is hydrolyzed to its constituent amino acid . The labeled amino acid is identified.

DANSYL CHLORIDE METHOD

DISADVANTAGE:•The hydrolysis stage destroys the polypeptide , these procedures cannot be used to sequence a polypeptide beyond its amino-terminal residue . ADVANTAGE:•It can help to determine the number of chemically distinct polypeptides in a protein , provided each has a different amino terminal residue.

EDMAN DEGRADATION

EDMAN DEGRADATION• The Edman degradation procedure labels and removes only the

amino terminal residue from a peptide , leaving all other peptide bonds intact. The peptide is reacted with phenylisothiocyanate under mildly alkaline conditions , which converts the amino terminal amino acid to a phenylthiocarbamoyl (PTC) adduct. The peptide bond next to the PTC adduct is then cleaved in a step carried out in anhydrous trifluoroacetic acid, with removal of the amino- terminal amino acid as an anilinothiazolinone derivative. The derivatized amino acid is extracted with organic solvents, converted to the more stable phenylthiohydantoin derivative by treatment with aqueous acid and then identified.

EDMAN DEGRADATION• After removal and identification of the amino terminal residue, the

new amino-terminal residue so exposed can be labeled, removed, and identified through the same series of reactions .This procedure is repeated until the entire sequence is determined.• The Edman degradation is carried out in a machine, called a

sequenator, that mixes reagents in the proper proportions, separates the products, identifies them, and records the results. • These methods are extremely sensitive. Often, the complete amino

acid sequence can be determined starting with only a few micrograms of protein.

SEQUENCING OF LARGE PROTEINSSEQUENCING OF LARGE PROTEINS

BREAKING DISULFIDE BONDS

CLEAVING POLYPEPTIDE CHAINS• Enzymes called proteases catalyze the hydrolytic cleavage of peptide

bondsREAGENT CLEAVAGE POINTS

trypsin Lys, Arg (C)

chymotrypsin Phe, Trp, Tyr(C)

Asp-N-protease Asp , Glu (C)

Pepsin Leu , Phe, Trp, Tyr (N)

Elastase Ala , Gly , Ser (C)

Cyanogen bromide Met (C)

Endoproteinase Lys C Lys (C)

SEQUENCING THE PEPTIDE• Each peptide fragment resulting from the action of protease is

sequenced separately by the Edman procedure.

ORDERING THE PEPTIDE FRAGMENTS

• Another sample of the intact polypeptide is cleaved into fragments using a different enzyme or reagent , one that cleaves peptide bonds at points other than those cleaved by first protease .• The amino acid sequences of each fragment obtained by the two

cleavage procedures are examined. • Overlapping fragments obtained from the second fragmentation yield

the correct order of the peptide fragments produced in the first.

QUESTION• TRYPSIN TREATMENT- CHYMOTRYPSIN TREATMENT

Leu–Glu Gln–Ala–Phe Gly–Tyr–Asn–Arg Asn-Arg-Leu-Glu Gln–Ala–Phe–Val–Lys Val-Lys-Gly-Tyr

ANSWERGln–Ala–PheGln-Ala-Phe-Val-Lys Val-Lys-Gly-Tyr Gly-Tyr-Asn-Arg Asn-Arg-Leu-Glu Leu-GluGln-Ala-Phe-Val-Lys-Gly-Tyr-Asn-Arg-Leu-Glu

MASS SPECTROMETRY• Mass spectrometry has emerged as an important technique for

characterizing and sequencing polypeptides.• Mass spectrometry accurately measures the mass to-charge (m/z)

ratio for ions in the gas phase (where m is the ion’s mass and z is its charge).

TANDEM MASS SPECTROMETRY• Used to sequence short stretches of polypeptide• A solution containing the protein under investigation is first treated with a

protease to hydrolyze it to a mixture of shorter peptides and the mixture is then injected into a device that is essentially two mass spectrometers in tandem.• The first mass spectrometer functions to select and separate the peptide ion of

interest from peptide ions of different masses as well as any contaminants that may be present.• The selected peptide ion is then passed into a collision cell, where it collides with

chemically inert atoms such as helium. The energy thereby imparted to the peptide ion causes it to fragment predominantly at only one of its several peptide bonds, thereby yielding one or two charged fragments per original ion.• The molecular masses of the numerous charged fragments so produced are then

determined by the second mass spectrometer.

• By comparing the molecular masses of successively larger members of a family of fragments, the molecular masses and therefore the identities of the corresponding amino acid residues can be determined.• The sequence of an entire polypeptide can thus be elucidated• Computerization of the mass-comparison process has reduced the

time required to sequence a short polypeptide to only a few minutes• Mass spectrometry can also be used to sequence peptides with

chemically blocked N-termini (which prevents Edman degradation) and to characterize other posttranslational modifications such as the addition of phosphate or carbohydrate groups