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 composition

Hydrolyse peptide heating in 6 N HCl at 110C 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 a-NH2 group to form a sulfonamide derivative Hydrolyse into dabsyl-peptide in 6 N HCl Identify 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 phenylthiohydantoin (PTH)-amino acid, Can be identified by chromatography The Edman procedure is repeated on the shortened peptide, yielding another PTHamino acid, a

This is again identified by chromatography

Sequencing: Edman degradation

Use HPLC to resolve PTH-amino acids

Edman degradation: Limitations

The 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: Solutions

Cleave peptide with proteases, such as trypsin, chymotrypsin, V8 protease Edman degradation fragments, to sequence

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 degradation

The 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 degradation

Chymotryptic 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 Blotting
Blotting 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 Blotting
The 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 Blotting
Other 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 TrisHCl/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 (Nitrocellulose, Nylon, difluoride; PVDF) 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. Membranes: polyvinylidene

Amido Black staining (not satisfactory);

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 (Sigma),

phosphate

(BCIP;

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 nonspecific 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 contd
Probing 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 buffer Add substrate: +10 ml/100 cm2 blot.

Procedure contd
Visualisation 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