Identification of a large biological molecule Identification of a large biological molecule

Protein sequencing
DNA sequencing
Protein sequencing
Step 1
DNA fingerprint (restriction map)
Protein fingerprint
Step 2 ?
Amino acid 1

DNA Protein
?
Step 3 Amino acid 2

Sanger’s method Edman Degradation

Protein degradation method developed by
Pehr Victor Edman 1916-1977

1. Coupling
2. Cleavage
3. Conversion
4. Chromatography

Step 1

Amino acid 1 Step 2

Amino acid 2 Step 3

Coupling Coupling
phenylisothiocyanate (PITC)

1
Coupling Cleavage

ATZ-amino acid

Conversion reverse-phase C-18 column for detection at 270nm

standard mixture of 19 PTH-amino acids

ATZ-amino acid
PTH-amino acid

Chromatography (HPLC)

PTH-amino acid
Standard

Standard

Residue 1
(Leucine)

2
Edman Degradation Identification of a large biological molecule
Protein degradation method developed by protein ladder sequencing
Pehr Victor Edman 1916-1977

1. Coupling Step 1
2. Cleavage
3. Conversion Step 2
4. Chromatography
Step 3
Step 1

Amino acid 1 Step 2

Amino acid 2 Step 3

Sanger Identification of a large biological molecule

DNA ladder sequencing

Chain termination
Ladder formation

Identification of a large biological molecule

Identification of a large biological molecule
DNA ladder sequencing Fingerprint (Pattern, Profile)
Specific cutting

A B C D

C
D

A
B

M/z
Electrophoresis
Mass spectrometry

3
 DNA fingerprint (Restriction map) restriction enzymes

The Nobel Prize in Physiology or Medicine 1978

Werner Arber Daniel Nathans Hamilton O. Smith

Arber discovered restriction enzymes.

Smith verified Arber's hypothesis.
Nathans pioneered the application of restriction enzymes to genetics.
EcoR I
5´ ...G^A A T T C ... 3´ 5´ ...G A A T T C ... 3´
3´... C T T A A^G ... 5´ 3´... C T T A A G ... 5´

restriction enzymes
Gene cloning

Restriction map Restriction map

(electrophoresis)

4
 Restriction map Restriction map
(electrophoresis vs MALDI-TOF)

- -
- +

Restriction map Restriction map

SV40 virus Human ?

亞當、夏娃

稅老師
ET

DNA Fingerprinting using RFLP Peptide Mass Fingerprint (PMF)

(Restriction Fragment Length Polymorphisms)

Software and computational techniques for the identification

of proteins and residue modifications using MALDI-TOF MS

Protein Database

5
Peptide Mass Fingerprint (PMF)
Gel
Sample preparation
Peptide Mass Fingerprint (PMF)
1. digestion of a protein by an enzyme provides a fingerprint of
great specificity
2. PMF is limited to the identification of proteins for which
sequences are already known

MALDI-TOF MS

Peptide mass
fingerprint

Peptide identification

Theoretical mass values http://www.matrixscience.com

(silico digestion of protein sequences)
Database

Experimental peptide masses

Peptide Mass Fingerprint (PMF) •Choice of Enzyme

• Choice of Enzyme
• Missed Cleavages Specific cleavage
• Masses tolerance
• Constraining the Protein Molecular Weight
• Which masses to include in a search
• Autolysis products
• Modifications Autolysis

6
 Choice of Enzyme (Specific cleavage) Modified Trypsin (TPCK-treated)

Modified Trypsin is treated with TPCK to inactivate any

remaining chymotryptic activity.

It is modified by acetylation of the ε-amino groups of

lysine residues to prevent autolysis.

Peptide Mass Fingerprint (PMF) Missed Cleavages

• Choice of Enzyme
• Missed Cleavages
• Masses tolerance
• Constraining the Protein Molecular Weight
• Which masses to include in a search
• Autolysis products
• Modifications

http://www.matrixscience.com Peptide Mass Fingerprint (PMF)

• Choice of Enzyme
• Missed Cleavages
• Masses tolerance (mass correction)
• Constraining the Protein Molecular Weight
• Which masses to include in a search
• Autolysis products
• Modifications

7
Mass correction Mass correction

Sample
Standard

Ez = (1/2) mv2

V = L/T

Mass correction Mass correction

V = L/T

V = L/T

Ez = (1/2) mv2
V = L/T V = L/T

External standard
Ez = (1/2) mv2
M/z

V = L/T
M/z M/z
V = L/T Standard mass Time*Time sample

Mass correction Mass correction

External standard External standard
M/z M/z

M/z M/z M/z M/z

Standard mass Time*Time sample Standard mass Time*Time sample

8
http://www.matrixscience.com Mass correction
Masses tolerance Internal standard
200 ppm

M/z
sample

Autolysis Mass correction

Low-level digests can be dominated by autolysis peaks.
External standard
internal mass calibration
M/z
porcine trypsin
M/z M/z
Standard mass Time*Time sample

Internal standard

M/z
sample

http://www.matrixscience.com Peptide Mass Fingerprint (PMF)

Masses tolerance
50 ppm • Choice of Enzyme
• Missed Cleavages
• Masses tolerance
• Constraining the Protein Molecular Weight
• Constraining the Protein pI
•Which masses to include in a search
• Autolysis products
• Modifications

9
Constraining the Protein Mw and pI http://www.matrixscience.com
Mw: 49.9 pI: 4.8 Mw: 49.9 pI: 4.8

Constraining the Protein Molecular Weight Problem: lower sequence coverage

Database usually contains the least processed form of a protein Insulin
Database 5734 Da

Pre-insulin Connecting
signal peptide
Pre-insulin Connecting
signal peptide 11,394 Da

11,394 Da

Mascot http://www.matrixscience.com
Mw: 49.9 pI: 4.8
protein molecular weight is applied as a sliding window
5734 Da

Connecting
signal peptide

11,394 Da

10
Peptide Mass Fingerprint (PMF) Alkylation

• Choice of Enzyme
• Missed Cleavages
• Masses tolerance
• Constraining the Protein Molecular Weight
• Which masses to include in a search
• Autolysis products
• Modifications

Alkylation Alkylation (iodoacetamide)

Problem: mass shift http://www.matrixscience.com

Insulin
5734 Da

Pre-insulin Connecting
signal peptide

11,394 Da

11
Peptide Mass Fingerprint (PMF) http://www.matrixscience.com

• Choice of Enzyme
• Missed Cleavages
• Masses tolerance
• Constraining the Protein Molecular Weight
• Which masses to include in a search
• Autolysis products
• Modifications

MALDI-TOF with Reflectron

C12, C13

M/z
sample

average

monoisotopic

http://www.matrixscience.com http://www.matrixscience.com

M/z
sample

12
Search results Search results

Search results Search results

Search results Search results

13