Transcript Lecture 6: Peptides

Lecture 6: Peptides
– MS-MS
– Schiff base for ninhydrin
– Protein purification
Figure 7-7
The amino acid sequence of a polypeptide chain.
To make trypsin even more versatile you
can modify side chains of amino acids
Lys specific reaction to hide basicity
See p. 170 in your book-especially the reactions with
citraconic anhydride so trypsin won’t cleave at Lys
residues.
Also on p. 170 conversion of Cys side group with 2bromoethylamine to make a basic group to cleave at
Cys with trypsin.
Lysine reactions
H
O
+
R’ C
H
-OOC
+
CH
CH
CH
NH
C CH2
2
2
2
3
+
H3N
Lysine
aldehyde
H
-OOC
O
C CH2 CH2 CH2 CH2 N C
+
H3N
+
H
Schiff base
H2O
+
H+
Lysine reactions
O
H2C C
O
H
+
-OOC
+
CH
CH
CH
NH
C CH2
2
2
2
3
+
H3N
H2C C
Lysine
O
Succinic
anhydride
H
C CH2 CH2 CH2 CH2 N C CH2 CH2 C
+
H3N
O
O
-OOC
O-
+ 2H+
Determining primary structure of
polypeptides
Deduce the amino acid sequence of a simple polypeptide
from the following results:
A. Acid hydrolysis: (Ala2, Arg, Lys2, Met, Phe, Ser2)
B. Carboxypeptidase A: (Ala)
C. Trypsin: (Ala,Arg), (Lys,Phe,Ser), (Lys), (Ala, Met, Ser)
D. CNBr: (Ala, Arg, Lys2, Met, Phe, Ser), (Ala, Ser)
E. Thermolysin: (Ala, Arg, Ser), (Ala, Lys2, Met, Phe, Ser)
Where do we start?
First, from A. (acid hydrolysis) we know how many amino
acids are in the polypeptide: 9
Second from B. (carboxypeptidase A), we know the last
amino acid is one of the Ala.
1 - 2 - 3 - 4 - 5 - 6 - 7 - 8 -Ala
We know trypsin cleaves at the carboxy side of basic aas
(Lys and Arg)
Trypsin: (Ala,Arg), (Lys,Phe,Ser), (Lys), (Ala, Met, Ser),
so we can rearrange the amino acids as follows:
Ala-Arg, either Phe-Ser-Lys or Ser-Phe-Lys, Arg-Lys or
Lys-Lys, and either Lys-(Ala, Met, Ser) or Arg-(Ala,
Met, Ser).
For CNBr, we got two fragments (Ala, Arg, Lys2, Met,
Phe, Ser) and (Ala, Ser). We know that cleavage
occurs on the carboxy side of Met. So we know that
Met-(Ser-Ala) or Met-(Ala-Ser).
1 - 2 - 3 - 4 - 5 - 6 - 7 - 8 -Ala
For thermolysin, we know it cleaves N-terminal to Ile,
Met, Phe, Trp, Tyr, Val. So (Ala, Arg, Ser) are before
Met
From trypsin: Ala-Arg, Phe-Ser-Lys or Ser-Phe-Lys, ArgLys or Lys-Lys, and either Lys-(Ala, Met, Ser) or Arg(Ala, Met, Ser).
WE know that one Ala is the carboxy terminal amino acid,
so Ala-Arg cannot be the carboxy terminus. Therefore,
the only other possibility is the last sequence (Ala, Met,
Ser) where Ala is the carboxy terminal amino acid. So
the order at the carboxy terminus is basic aa-Met-SerAla or basic aa-Ser-Met-Ala
For CNBr, we know that cleavage occurs on the carboxy
side of Met. So, combined with the trypsin result we
get basic aa-Met (Ser-Ala).
1 - 2 - 3 - 4 - 5 - basic aa - Met - Ser -Ala
For thermolysin, we know it cleaves N-terminal to Ile,
Met, Phe, Trp, Tyr, Val. So (Ala, Arg, Ser) are before
Met or Phe.
We know from the CNBr cleavage that the Met must be
before Ser-Ala, so for the (Ala, Lys2, Met, Phe, Ser)
Phe must be the 1st aa in this sequence. We also
know that a basic aa precedes Met from the trypsin
experiment. Since the only basic aas in this fragment
are Lys, the order must be : Phe-Lys-Lys-Met-Ser-Ala
1 - 2 - 3 - Phe - Lys - Lys - Met - Ser -Ala
Remember for thermolysin, we know it cleaves N-terminal
to Ile, Met, Phe, Trp, Tyr, Val. So (Ala, Arg, Ser) are
before Met or Phe.
We know from the trypsin digest that Ala-Arg are in a
specified order so the final sequence must be Ala-ArgSer
Ala - Arg - Ser - Phe - Lys - Lys - Met - Ser -Ala
Peptide characterization by mass
spectrometry
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Mass spectrometry (MS) is an important technique for characterizing and sequencing
polypeptides.
MS measure the mass-to-charge (m/z) ratio for ions in the gas phase where m is the
mass of th ion and z is the charge.
Prior to 1985 MS could not be used for the analysis of proteins or nucleic acids
because the gas phase ions produced destroyed the molecules.
Three techniques developed to solve this problem:
– Electrospray ionization (ESI)
– Matix-assisted laser desorption/ionization (MALDI)
– Fast atom bombardment (FAB).
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Figure 7-8a
The generation of the gas phase ions required for
the mass spectrometric analysis of proteins. (a) By electrospray
ionization (ESI).
Page 172
Figure 7-8b
The generation of the gas phase ions required for
the mass spectrometric analysis of proteins. (b) By matrix-assisted
laser desorption/ionization (MALDI).
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Figure 7-8c
The generation of the gas phase ions required for
the mass spectrometric analysis of proteins. (c) By fast atom
bombardment (FAB).
p1= M + z1
z1
p2= M + z1-1
z1-1
Z1 = 25 for peak 1
P1 = 678.6
The mass of = 678.6 (25)-25=16940
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1884.7(9)-9=1695.33
Figure 7-9
The ESI-MS spectrum of the 16,951-D horse heart
protein apomyoglobin.
Peptide characterization by mass
spectrometry
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Gas phase macromolecular ions are detected by MS which measures their m/z
values with high accuracy >0.01%.
If the ion’s charge value can be determined, it is the most accurate method.
ESI-MS has several advantages over other forms of MS because of the high ionic
charges of the ions produced allows analysis of compounds with greater molecular
mass >100 k.
ESI-MS can be configured with other purification equipment (HPLC for example) so
that it can be used to analyze a tryptic digest of a protein by determining the
molecular masses of the components.
Tandem mass spectrometers (MS-MS) can be used to directly sequence short
polypeptides (<25 aa) but cannot distinguish Ile and Leu (same mass) and is
unreliable for Gln and Lys (differ by only 0.036 D)
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Figure 7-10
The use of a tandem mass spectrometer (MS/MS)
in amino acid sequencing.
Carboxyl and amino terminal reactions
are important for chemical synthesis of
peptides
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Polypeptides can be chemically synthesized by covalently linking (coupling)
amino acids, one at a time, to the terminus of a growing polypeptide chain.
In the chemical synthesis of polypeptides, the polypeptide chain is being
synthesized from the C-terminus to the N-terminus.
The amino acid being added to the chain must already have its own amino group chemically protected (blocked) to prevent it from reacting with
other similar molecules as well as with the N-terminal amino group of the
chain.
Once the new amino acid is coupled, the now N-terminal amino acid must
be deprotected (deblocked) so that the next peptide bond can be formed.
Figure 7-34 Flow diagram for polypeptide synthesis by
the solid phase method.
Amino group reactions
R
H
C
NH2
COOAmino acid
O
+
R’
C
H
Cl
HCl
R
H
C
N
COO-
C-R’ + H+
O
Substituted
amide
Protection of the amino group
O
(CH3)3C-O
C
R
+
Cl
t-Butyloxycarbonyl chloride
H2N
COO-
H
(Boc)
-amino acid
HCl
R
O
(CH3)3C-O
C
C
NH
C
H
Boc-amino acid
COO-
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Figure 7-35
A selection of amino acids with benzyl-protected
side chains and a Boc-protected -amino acid group.
Carboxyl group reactions
+
NH3
R
C
+
H3N
COOH + NH3
H
R
C
O
NH2
C
H
H2O
Amide
Amino acid
+
NH3
R
C
+
H3N
COOH + R’-NH2
H
Amino acid
R
H2O
C
H
O
C
N-R’
H
Substituted
amide
Carboxyl group reactions
+
NH3
R
C
+
H3N
COOH + R’-OH
H
Amino acid
R
H2O
C
H
O
C
O-R’
Ester
Protection of the carboxyl groups
+
NH3
R
C
+
H3N
COOH + NH3
H
R
C
O
NH2
C
H
H2O
Amide
Amino acid
+
NH3
R
C
+
H3N
COOH + R’-NH2
H
Amino acid
R
H2O
C
H
O
C
N-R’
H
Substituted
amide
Amino group reactions
R
C
H
R
Primary amine
O
NH2 +
COO-
R’
C
H
H
O
NH2 +
COOAmino acid
C-R’ + H+
H
Schiff base
R
C
N
COO-
H2O
Amino acid
H
C
imine
R’
C
H
Cl
HCl
R
H
C
N
COO-
C-R’ + H+
O
Substituted
amide
Example of the Schiff base reaction
using Ninhydrin
O
O
OH
OH

H2O
O
Ninhydrin
O
OH
NH
O
COOO +
H2N
H
R
O
Proton transfer
C
O
ONH+
2
COOO
R
R
COO-
Ninhydrin reaction
O
O
OH
NH
COO-
O
O

H2O
N
O
C-OR
R
O
O
H
H
H+
O
: :
O
N
N
+ CO2
H
R
O-
:
O-
R
O
H
H
Ninhydrin reaction
O
O
H
O
H
H H+
H
H
OH
: :
:
N
O
N
H
O-
O
O-
R
R
O
O
H
+
N
NH2
O- H
O+
H
+
R
OH
+
OH
H
R
OH
R
O
H
R
Ninhydrin reaction
O
O
NH2
+

O
O
O-
Imine formation followed by same mechanism
O
O
N
H
N
O
O
O
O-
O
O
Two resonance forms of Ruheman’s Purple can be detected at 570 nm
Summary of reactions of amino acids
• Peptide bond formation
– Activation
– Protection
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Schiff’s base formation
Sanger’s reagent
Dansylation
Edman degradation
Peptide hydrolysis
Hydrolysis of Gln, Asn
Modification of Lys
Reactions of Cys (-mercaptoethanol, DTT, iodoacetate)
Met/CNBr
Hydrazine Cleavage
Protein purification
• Protein purification can be thought of as a series of fractionation
steps designed to:
– Get the protein of interest almost exclusively in one fraction
– Get a significant amount of contaminants in a different fraction
• Objective: To separate the protein of interest from a complex
mixture of proteins (from tissues or recombinant organism) while
maintaining biological function.
• Biological function can be maintained by controlling the pH, the
temperature, and ionic strength (e.g. salt concentration) of the
buffers used during purification.
• Similar techniques are used to isolate all proteins but the less
starting material we have the more work it is to isolate it.
Outline of steps for protein purification
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Choose your protein source.
Methods of solubilization
A unique assay (a test) for the protein of interest.
Assay for total protein
A series of fractionation steps adapted to the protein
Assays for purity
• If the protein you want to purify is an enzyme, it’s biological activity
can be used as a unique assay for its presence throughout the
purification.
Starting materials for protein purification
• Proteins constitute a major fraction of the mass of all living things
• Sometimes proteins are available in abundance (hemoglobin) but
they may also be very scarce (lac repressor in wild type E. coli
cells).
• Therefore, the choice of where proteins are obtained is important.
• If more of the protein is present in the source, the easier it is to
purify.
• Proteins may be obtained from an original source (tissues from
specific organism, yeast, etc.)
• Proteins may also be obtained from recombinant organisms
(clones) that overexpress the gene encoding the protein of interest.
• In E. coli and yeast recombinant protein may constitute up to 30% of
the total protein.
Methods of solubilization
• 1st step is to get the protein into solution
• If the protein is located in the cytosol, we need to break open the
cell.
• For animal cells this can be accomplished with osmotic lysis. Put
the cells in hypotonic solution; less solutes than inside the cell.
• For cells with a cell wall (bacteria, plants) we have to use other
methods.
• For bacteria, lysozyme is often effective - Selectively degrades
bacterial cell wall.
• Can also use detergents or organic solvents, although these may
also denature the protein.
Mechanical processes to break open the
cell
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High speed blender
Homogenzier
French press
Sonicator.
After the cells have been broken, the crude lysate, may be filtered or
centrifuged to remove the particulate cell debris. The protein of
interest is in the supernatant.
For proteins that are components of
membranes or subcellular assembly
• Remove the assembly from the rest of the cellular material
(mitochondria for example).
• Can be done by differential centrifugation-cell lysate is centrifuged
at a speed that removes only the cell components denser than the
desired organelle followed by a centrifugation at speed that spins
down the organelle.
Stabilization of proteins
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pH
Temperature
Inhibition of proteases
Retardation of microbes that can destroy proteins
– Sodium azide is often used.
Assay of proteins
• Done throughout the purification process to make sure that your
protein of interest is there.
• If the protein of interest is an enzyme, using a reaction for which that
enzyme is a catalyst is a good way to monitor protein recovery.
• Monitor the increase of the product of the enzymatic reaction
– Fluorescence
– Generation of acid to be monitored by titration
– Coupled enzymatic reaction - couple with another enzyme to make
an observable substance.