Protein sequencing

By-
Vikas Kr. Singh, M. Sc. Biotechnology
vikasbiotech10@gmail.com

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

Strategy for protein sequencing
• 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.

End-group Analysis
• Number of chains can be determine by
identifying the number of N- and C-terminal.
• N-terminal analysis
– Dansyl chloride or FDNB method
– Phenylisothiocynate (PITC)/ Edman reagent
– Aminopeptidase
• C-terminal analysis
– carboxypeptidase

N-terminal Analysis with Dansyl Chloride
• Reagent: 1-dimethyl
aminophthalene-5-sulfonyl
chloride (dansyl chloride)
• Dansyl polypeptide 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.vikasbiotech10@gmail.com

N-terminal Analysis Edman (Degradation)
• Nucleophilic attack on
phenyl isothiocyanate
(PITC), the Edman
reagent, under mild
alkaline conditions
(Nmethylpiperidine/
water/ methanol).
• Formation of a
phenylthiocarbamyl
derivative (PTC-
peptide)

• Anhydrous trifluoro
acetic acid (TFA) 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.

• 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

• 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).

How to overcome with the
by-products formed in Edman
degradation without losing
the polypeptide chain?

Solid-phase matrix-the Merrifield resin

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.
• Further analyzed by
amino acid analyzer.

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

Separation and Molecular Weight
Determination of Subunits
• Traditional Methods:-
– SDS-PAGE, SEC, or RP-HPLC used to separate
the subunits after cleavage of disulfide
bonds.
– Mw standards and a calibration curve are used
to determine the Mw.
– The approximate no. of amino acids estimated
from the Mw of the subunit using 110 Da as
the average molar mass for each amino acid.
• Recent methods
– MALDI:- more accurate and fast.vikasbiotech10@gmail.com

Amino Acid composition
• Strategy:-
-Hydrolysis followed by separation and identification
• Acid catalyzed hydrolysis
-6M HCl/ 100-120°C/ 24 h (in oxygen free environment
to prevent oxidation of SH groups)
-Some residues are degraded under these harsh
conditions
• Base catalyzed hydrolysis
-4 M NaOH /100°C/ 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

• 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
Amino Acid composition

Cleavage of Specific Peptide Bonds
• Direct sequencing is applicable to peptides that
have up to about 50 residues only.
• Problems occur after lengthy reactions
-Incomplete reactions
-Accumulation of impurities from side reactions
• Solution:- Use enzymes to fragment the
polypeptide chain.
-Proteolytic enzymes: - endopeptidases and
- exopeptidases.

Enzymatic Fragmentation

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

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.

Protein sequencing

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