PROTEIN SEQUENCING. First Sequence

PROTEIN SEQUENCING

First Sequence

• The first protein sequencing was achieved by Frederic Sanger in 1953. He

determined the amino acid sequence of bovine insulin

• Sanger was awarded the Nobel Prize in

1958

I. Strategy

• Determine number of polypeptide chains (subunits)

• Determine number of disulfide bonds (inter- and intra- chain)

• Determine the amino acid composition of each polypeptide chain

• If subunits are too large, fragment them into shorter polypeptide chains

• Sequence each fragment using the Edman degradation method

• Complete the sequence by comparing overlaps of different sets of fragments

II. End-group Analysis

• Number of chains can be determine by identifying the number of N- and C-terminal.

• N-terminal analysis

– Phenylisothiocynate (PITC)/ Edman reagent – Aminopeptidase

• C-terminal analysis

N-terminal Analysis with Dansyl Chloride

• Main reagent: 1-dimethyl aminophthalene-5-sulfonyl chloride (dansyl chloride)

• Dansyl poplypeptide chain is prepared

• Acidic hydrolysis liberates all amino acid and the N- terminal dansyl amino acid

• Amino acids are separated

• Fluorescence of the dansyl amino acid is detected

• Type of aa is obtained from comparison with standard dansylated amino acids

N-terminal Analysis Edman (Degradation)

• Nucleophilic attack on phenyl isothiocyanate (PITC), the Edman reagent, under mild alkaline conditions (N- methylpiperidine/water/

methanol)

• Formation of a phenylthiocarbamyl derivative (PTC-peptide)

N-terminal Analysis Edman (Degradation)

• Anhydrous trifluoro acetic acid (TFA) is used to cleave the terminal amino acid in the form of a thiozolinone derivative leaving the other peptide bonds intact

• The thiozolinone (TZ) derivative is extracted in an organic solvent (e.g.

N-butyl chloride)

• Peptide cleaved carries a free amino terminus

N-terminal Analysis Edman (Degradation)

• The TZ is extracted into an organic solvent and treated with an acid (25 % TFA/water) to form phenylthiohydantoin (PTH)

derivative

• PTH is detected from UV absorption at 296 nm

N-terminal Analysis-Edman Degradation

• PTH amino acid is separated from the other components by chromatography or electrophoresis

• The terminal amino is identified according to retention time or mass

• This sequence can be repeated to identify all amino acid in short peptide chains (40-60 amino acid long)

Edman Degradation on Protein Sequencer

Perkin Elmer Applied Biosystems Model 494 Procise protein/peptide sequencer http://www.biotech.iastate.edu/facilities/protein/nsequence494.html

Edman Degradation on Protein Sequencer

By-products of Edman Degradation

N- and C-terminal Analysis-Exopeptidase Method

• Exopeptidases cleave the terminal residue of a polypeptide chain

• Aminopeptidases cleave the N-terminal residues

• Carboxypeptidases cleave the C-terminal residues

• Aminopeptidases and carboxypeptidases are highly specific, thus are of limited use due to slow rates and resistance of some amino to cleavage

III. Disulfide Bond Cleavage

• Disulfides are reduced to thiol with dithiothreitol (DTT) or 2-

mercaptoethanol

• Thiols are treated with alkylating agents (e.g.

iodoacetic acid) to

prevent the re-oxidation

during subsequent

steps.

Protection of sulfyhydryl groups

IV. Separation and Molecular Weight Determination of Subunits

• Traditional Methods

– SDS-PAGE, SEC, or RP-HPLC are used to separate the subunits after cleavage of disulfide bonds

– Mw standards and a calibration curve are used to determine the molecular weights

– The approximate number of amino acids can be estimated from the Mw of the subunit using 110 Da as

V. Amino Acid composition

• Strategy:

– hydrolysis followed by separation and identification

• Acid catalyzed hydrolysis

– 6M HCl/ 100-120ºC/ 24 h (in oxygen free environment to prevent oxidation of SH groups)

– Some residues are degrated under these harsh conditions

• Base catalyzed hydrolysis – 4 M NaOH /100ºC/ 4-8 hours

– Arg, Cys, Ser and Thr are decomposed and other amino acids are deaminated and racemized

– Used mainly to determine Trp which is extensively degraded under acid catalyzed hydrolysis

V. Amino Acid composition

• Enzymatic hydrolysis

– By exo- and endopeptidases

– A combination of endo and exopeptidases must be used to hydrolyze all the peptide bonds

• Separation

– Individual amino acids in hydrolyzed mixture can be separated by RP-HPLC or CE and identified

according to retention time

• Increasing sensitivity

– Pre- or post-column derivatization is used to increase sensitivity

Derivatization with OPA and MCE

VI. Cleavage of Specific Peptide Bonds

• Direct sequencing is applicable to peptides that

• Problems which occur after lengthy reactions – Incomplete reactions

– Accumulation of impurities from side reactions

• Solution: use enzymes to break down the

polypeptide chain into shorter fragments

Enzymatic Fragmentation

• Trypsin

– Trypsin is the most commonly used proteolytic

enzyme. It cleaves at the C-end of positively charged amino acids (Arg and Lys) if the next residue is not a proline.

– It is highly specific

– Cleavage sites may be removed or added via derivatization to take advantage of the specificity of trypsin

– Reaction times can be adjusted to limit proteolysis if there are too many Arg and Lys residues

– Non-denaturing conditions can be used to limit proteolysis as well

Trypsin Digestion

Derivatization of Cys for Tryptic Digestion

Other Proteolytic Enzymes

• Endopeptidases

– Pepsin; cleaves at the amino end of Phe, Tyr, Trp the previous residue is not a proline

– Chymotrypsin: cleaves at the carboxyl end of Phe, Trp, Tyr if the next residue is not proline

– Endopeptidae GluC: cleaves at the carboxy end of Glu

• Exopeptidases

– Leucine aminopeptidase: cleaves rapidly N-terminal leucine aa.

Does not cleave N-terminal proline

– Aminopeptidase M: cleaves all N-terminal residues – Carboxypeptidase A: cleaves all except Arg, Lys, and Pro

• Especially efficient for aa with bulky aliphatic and aromatic side

Chemical Fragmentation Methods

• Cyanogen bromide (CNBr) specifically cleaves Met residues at the C-end forming a homoserine lactone

1.

2.

3.

4.

Sequence Determination

• Separate segments by chromatography or

electrophoresis and sequence fragments individually

• Edman degradation is the method of choice

– Fully automated systems which use the Edman degradation methods are available commercially (Sequenator)

• In the sequenator the protein is immobilized through bonding to a solid support or by adsorbing it onto an inert glass frit.

• Controlled amounts of reagents are injected by a pumping system

• The thiozolinone is transferred to a conversion chamber for hydrolysis to the PTH amino acid

• The final product, the PTH amino acid, is pumped into an HPLC column for on-line analysis

– 1 hour analysis time is possible for 50 amino acid residues

The solid-phase matrix-the Merrifield resin

Edman degradation

Ordering of Peptide Fragments

• Compare amino acid sequence of one set of peptide fragments with the sequence of a second set of fragments obtained using different cleavage points

Determination of Disulfide Bond Position

• Digest polypeptide chain(s)

• Run 2D gel of mixture of fragments using same conditions in both dimension

• After separation in the first dimension, the matrix is exposed to performic acid which cleaves all possible disulfide bonds

• Separation in the second dimension is performed

– Fragment without ss bonds will be positioned along the diagonal of the matrix

– Fragments linked by S-S bonds will produce off diagonal spots – The disulfide linked fragments can be extracted from the gel and

sequenced

Protein Sequencing by Mass Spectrometry

• Digest protein

• Obtain MALD TOF mass spectrum of digest

• Use online database to match fragments patterns with those in the data base

• Obtain sequence of fragments by performing MS/MS