Protein sequencing

The next step after the protein of interest has

been purified and its molecular weight

determined.

To get information about

Protein's function

Evolutionary history

First step is to determine the amino acid

composition of the peptide

Amino acid compositionHydrolyse peptide heating in 6 N HCl at

110°C for 24 hours..

Ion-exchange chromatography on

sulfonated polystyrene columns to separate

the hydrolysates

The amino acid is identified by its elution

volume

Amino acid composition Quantify amino acid by reaction with

Ninhydrin, detection limit 1g (10nmol); or

Fluorescamine, lower limit 1ng(10pmol).

Amino acids heated with ninhydrin yield

intense blue color

Except for proline, which gives a yellow

color

Amino acid composition (contd)Amino acid concn, after heating with ninhydrin, is proportional to the optical absorbance.

Composition: ASP, 2 x Gly, Ala, Phe, Arg

Then determine N-terminal Amino acid Label with Dabsyl chloride to yield fluorescent derivatives that can be detected with high sensitivity. Dabsyl chloride reacts with an uncharged -NH2 group to form a sulfonamide derivative

Hydrolyse into dabsyl-peptide in 6 N HClIdentify the dabsyl-amino acid, by its chromatographic properties. Dansyl chloride forms fluorescent sulfonamides too!

Sequencing: Edman degradation The dabsyl method leaves behind a totally

degraded peptide. In the Edman degradation,Phenyl isothiocyanate reacts with the

uncharged terminal amino group to form a phenylthiocarbamoyl derivative

Under mildly acidic conditions, a cyclic derivative of the terminal amino acid is liberated,

To leave an intact peptide shortened by one amino acid.

Sequencing: Edman degradation

The cyclic compound is a

phenylthiohydantoin (PTH)-amino acid,

Can be identified by chromatography

The Edman procedure is repeated on the

shortened peptide, yielding another PTH-

amino acid,

This is again identified by chromatography

Sequencing: Edman degradation

Use HPLC to resolve PTH-amino acids

Edman degradation: LimitationsThe efficiency of PTH-amino acid release

declines with number of cycles

Upper limit for this method is 50 residues

Most proteins are N-terminally

blocked e.g with an acetyl group

So cannot be sequenced using the

Edman degradation methodology.

Edman degradation: SolutionsCleave peptide with proteases, such as trypsin, chymotrypsin, V8 protease

Edman degradation to sequence fragments,

Use Dabsyl labeling to determine the N-terminal segments of the protein

Use of BLAST or other database searching programs to identify the intact protein.

Sequencing: Edman degradationThe Specific cleavage can be achieved by chemical or enzymatic methods. Cyanogen bromide (CNBr) splits polypeptide chains only on the carboxyl side of methionine residues

Sequencing: Edman degradation

Trypsin cleaves polypeptide chains on the

carboxyl side of arginine and lysine residues

Sequencing: Edman degradation

The peptides from specific cleavage are

separated by chromatography.

The sequence of each purified peptide

determined by the Edman method.

BUT

At this point, the order of the sequenced

segments is not defined.

Edman degradation: Order the peptides to

obtain the primary structure of the protein

The necessary additional information is

obtained from overlap peptides

Use another enzyme to split the polypeptide

chain at different linkages.

E.g. chymotrypsin cleaves preferentially on

the carboxyl side of aromatic and some other

bulky non-polar residues.

Sequencing: Edman degradationChymotryptic peptides overlap two or more tryptic peptides, Can therefore be used to assemble the entire amino acid sequence

Information obtained from Sequencing:

Infer the protein family/function by a BLAST

search

Establish evolutionary relationship:

Compare sequences of same protein from

different species

Presence of signal sequence as an

indicator of protein location e.g secretion

signal

Information obtained from Sequencing:

Sequence data provides a basis for

preparing antibodies specific for a protein of

interest.

In reverse genetics: DNA probes

corresponding to the amino acid sequence

can be constructed on the basis of the

genetic code.

Western BlottingBlotting involves transfer of separated proteins

onto nylon, nitrocellulose, or PVDF

membrane.

Why blot?

To make the protein more accessible to

probes,

Specific antibodies are preferred probes

Polyacrylamide is not particularly amenable

to the diffusion of large molecules.

Western BlottingThe attachment of specific antibodies to the

immobilised antigens can be readily

visualised by indirect enzyme

immunoassay techniques,

Usually a chromogenic substrate which

produces an insoluble product

Western BlottingOther types of probes include

Fluorescent labels (e.g. fluorescein)

Radioisotope labels (e.g. 125I)

Chemiluminescent substrates (highly

sensitive!)

Western Blotting: Antigen (blot) + antibodies

Then use secondary probes to detect the antibody binding

Conjugated anti-immunoglobulins (eg:

goat-anti-rabbit / human);

conjugated staphylococcal Protein A

( binds IgG of various species of animal); or

probes to biotinylated / igoxigeninylated

primary antibodies (eg: conjugated avidin /

streptavidin / antibody).

Western Blotting

During electroblotting,

Freshly-electrophoresed SDS-PAGE gels

are dipped into transfer buffer (0.025 M Tris-

HCl/20% (v/v) methanol, pH 8.3),

then laid flat on pre- wetted nitrocellulose

paper supported on three layers of wetted

filter paper resting on the ANODE

Western Blotting

Staining Proteins on Membranes:

(Nitrocellulose, Nylon, polyvinylidene

difluoride; PVDF)

Amido Black staining (not satisfactory);

India ink staining (OK),

Ponceau S (2 g Ponceau S + 30 g

trichloroacetic acid + 30 g sulfosalicyclic acid

+ water to 100 ml.) is highly useful.

Western Blotting: The Indirect enzyme

immunoassay,

Buffers and Solutions: Incubation/Blocking Buffer: 10 mM Tris-Cl/150

mM NaCl containing 1-5% non- fat milk powder and

0.05% Tween-20 or Triton X-100 (=Nonidet P-40),

pH 7.4.

Washing buffer: 10 mM Tris-Cl/150 mM NaCl pH

7.4 (OR SALINE) containing 0.05% Tween-20 or

Triton X-100.

Western Blotting: The Indirect enzyme immunoassay,

Buffers and Solutions contd: Substrate buffer: 100 mM Tris-Cl/100 mM NaCl/5 mM MgCl2 pH 9.5. Substrate stocks: Nitro Blue tetrazolium (NBT; Sigma), 75 mg/ml in 70% dimethyl formamide; 5-Bromo-4-chloro-3-indolyl phosphate (BCIP; (Sigma), 50 mg/ml in formamide (100%).

SUBSTRATE STOCKS IN 10 ML ALIQUOTS AT -20oC

Western Blotting: The Indirect enzyme immunoassay,Procedure:

Blocking: nitrocellulose blots are briefly rinsed in transfer buffer, then soaked for 1 hr at 37oC, or 2 hr at room temperature in blocking buffer. This procedure allows saturation of all non-specific protein binding sites on the blots.

Western Blotting: The Indirect enzyme immunoassay, Procedure contd

Attachment of specific antibodies to proteins: incubate blots in rabbit anti-sera diluted 1/10- 1/1000 in incubation buffer, in sealed boxes for 1 hr at room temperature on a shaking waterbath.

Washing: blots are washed by shaking in +100 ml/wash for 3x5 min., at room temperature.

Western Blotting: The Indirect enzyme

immunoassay, Procedure contdProbing of antibody binding:

is by means of suitably diluted alkaline

phosphatase - goat anti-rabbit globulins in

incubation buffer (eg 1/5000).

Incubation conditions and washing are the

same as for rabbit antiserum.

Western Blotting: The Indirect enzyme immunoassay, Procedure contd

Visualisation of Antibody binding to blots

Mix 50 ul of NBT stock with 10 ml of substrate buffer, then add 50 ul of BCIP stock and mix well by swirling.

Pre-rinse blot(s) in a little substrate bufferAdd substrate: +10 ml/100 cm2 blot.

Procedure contdVisualisation of Antibody binding to blots

Incubate in the dark with occasional agitation (30-60 min; Leave overnight with gentle agitation IN THE DARK if reactions are very faint.

Terminate reaction by washing with water.Blots are dried under weighted filter paper,

and stored in the dark. Photograph using a red filter for B& W, or

with no filter for colour slides.

500mA for 20-30 min to effect transfer.

Principle of chemiluminescence detection