Bio 98 - Lecture 5
Protein Sequencing &
Identification
Protein Identification
I.
Why?
• Find proteins associated with cancer and
other disease states
• Study protein modifications and interactions
II. Typical approach
1. 2D gel electrophoresis - very high resolution
separation of complex protein mixtures
2. Digest protein of interest with protease
3. Determine sequences or masses of the resulting
peptides
4. Deduce sequence or match masses to parent
protein
Proteins in a bacterial proteome
2D gel
pH 3
isoelectric point
pH 12
200 kDa
molecular weight
10 kDa
Proteins: E. coli: ~4,000; H. sapiens: ~20,000
2D gel electrophoresis
Proteins in a bacterial proteome
2D gel
pH 3
isoelectric point
pH 12
200 kDa
molecular weight
10 kDa
Proteins: E. coli: ~4,000; H. sapiens: ~20,000
Sequencing Proteins: A Stepwise Overview
I.
II.
III.
IV.
V.
Break and then block disulfide bonds.
Cleave protein into smaller peptides.
Separate the peptides.
Sequence the peptides.
Align the peptide sequences.
Insulin processing
insulin
I.
Breaking and blocking disulfide bonds
oxidation
reduction
Most common method: reduction & alkylation (next slides)
(i) Reduction step
BME
--Cys-|
S
|
S
|
--Cys--
2 R-SH
2(beta)-mercaptoethanol
or dithiothreitol (DTT)
--Cys—
|
SH
+
SH
|
--Cys--
R
|
S
|
S
|
R
(ii) Blocking step - alkylation
=
O
--Cys-|
SH
+
I-CH2-C-Oiodoacetic acid
=
--Cys-O
|
S-CH2-C-Ocarboxylmethylcysteine (CMC)
+
HI
II. Cleavage - using enzymes or chemicals
Enzymatic cleavage into peptide fragments using protease enzymes
O
O
---NH-CH-C-NH-CH-C--R1
R2
Protease X + H2O
O
---NH-CH-C-O +
R1
O
NH3-CH-C--R2
+
B. Reagents for selective cleavage of peptide bonds
Protease (source)
Specificity (N/C)
Trypsin (bovine pancrease)
Chymotrypsin (bovine pancrease)
Staphylococcus V8 protease
Pepsin (porcine pancrease)
Cyanogen bromide (chemical)
Lys, Arg (C)
Phe, Trp, Tyr (C)
Glu, Asp (C)
Phe, Trp, Tyr (N)
Met (C)
Peptide cleaved by Trypsin
N-terminus
Met-Leu-Trp-Ala-Cys-Lys-Ala-Gly
C-terminus
Peptide cleaved by Pepsin
N-terminus
Met-Leu-Trp-Ala-Cys-Lys-Ala-Gly
C-terminus
III. Separation & purification of peptides
HPLC: high-performance liquid chromatography
1. A form of liquid chromatography (LC) with small column
size, small “beads” inside column and high pressure.
2. Typically uses “reversed-phase” columns for separating
peptides.
• analogous to ion-exchange, except binding of protein to
bead is via hydrophobic interaction, not charge.
• elute peptides from “reversed-phase” beads by
increasing organic solvent, typically methanol or
acetonitrile, instead of salt.
hydrophobicity of peptides
% acetonitrile (------)
absorbance at 214 nm ( _____ )
Peptide separation by HPLC
Elution time (mins) 
IVa. Sequencing a polypeptide via Edman degradation
Phenyl-iso-thio-cyanate
N=C=S
PITC
+
polypeptide
N
•
PTH-AA1
Phenylthio-hydantoin
derivative
N
•
C
shortened polypeptide C
•
repeat the cycle
identification by HPLC
IVa2. Scheme of Edman degradation
IVa3. Complications with Edman degradation:
Blocked N-terminus
fMet-Ala-Ser-Glu-…
V. Aligning the peptides to establish final sequence
Trypsin cuts C-term of Lys (K), Arg (R)
Cyanogen bromide cuts C-term of Met (M)
IVb. Sequencing a polypeptide via
mass spectrometry (MS)
Spectral Output
Tandem MS or MS/MS
2D gel electrophoresis
2D gel electrophoresis
Peptide Mass Fingerprinting (PMF)
1. Cut out the gel spot containing your protein of interest and digest
it with a protease such as trypsin.
2. Then submit the entire mixture of peptides to mass spectrometry.
Tell computer
1. cleavage agent used
2. list of peptide masses
Get from computer
List of proteins that could have
produced those peptides