Download 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
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I. Strategy
• Determine number of polypeptide chains (subunits)
• Determine number of disulfide bonds (inter- and intrachain)
• 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
–
–
–
•
Dansyl chloride
Phenylisothiocynate (PITC)/ Edman reagent
Aminopeptidase
C-terminal analysis
–
carboxypeptidase
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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 Nterminal 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 (Nmethylpiperidine/water/
methanol)
• Formation of a
phenylthiocarbamyl
derivative (PTC-peptide)
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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
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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
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Edman Degradation on Protein Sequencer
By-products of Edman Degradation
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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 2mercaptoethanol
• Thiols are treated with
alkylating agents (e.g.
iodoacetic acid) to
prevent the re-oxidation
during subsequent
steps.
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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
the average molar mass for each amino acid
• Recent methods
– MALDI: more accurate and fast
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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
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Derivatization with OPA and MCE
VI. Cleavage of Specific Peptide Bonds
• Direct sequencing is applicable to peptides that
have up to about 50 residues only.
• 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
– Proteolytic enzymes: endopeptidases and
exopeptidases
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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
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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
chains
• Does not cleave if the next residue is Pro
– Carboxypeptidase B: cleaves C-terminal Arg and Lys if the next
residue is not Pro
– Carboxypeptidase C: cleaves C-terminal residues
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Chemical Fragmentation Methods
• Cyanogen bromide (CNBr) specifically
cleaves Met residues at the C-end forming
a homoserine lactone
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3.
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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
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The solid-phase matrix-the Merrifield resin
Edman degradation
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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
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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
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