Peptide mass fingerprinting is a technique to identify proteins by breaking them into peptides via enzymatic digestion, measuring the peptide masses using mass spectrometry, and comparing the results to theoretical peptide masses from protein sequence databases to find a match. The key steps are isolating the protein, digesting it into peptides, using MALDI or ESI mass spectrometry to determine peptide masses, running an in silico digestion of protein databases to generate theoretical peptide masses, and comparing the experimental and theoretical masses to identify the protein.

1. Protein Identification
Peptide Mass Fingerprinting

2. Protein fragment often generated by trypsin
….
The molecular size of peptides
Uniqueness
Mass
Peptide Fingerprinting
Peptide
Mass
Fingerprinting

3. Introduction
About Peptide Mass Fingerprinting (PMF)
• The method was developed in 1993
• A high throughput protein
identification technique in which the
mass of an unknown protein can be
determined
• Usually with a mass spectrometer such
as MALDI-TOF or ESI-TOF
….

4. The Principles
Is identical to
In Gel
Digestion
MS
1193.8
845
545.6
887.6
2383.7
In Silico Digestion
Database
1433.8
805.3
3005.3
702.4
988.7
183.9
2133.5
……
1193.7
845.3
545.8
887.8
2383.1
308.5
995
3100.4
Protein sequence
database
Theoretical
peptide masses
In silico
Digestion
Peptide match
Trypic
peptides
MS
Peptide
masses
….

5. 1.Protein Separation
Isolation of intact protein by 2D-PAGE. 2. Digestion
Digestion of protein into small
peptides by proteolytic treatment.
Trypsin is the favored enzyme for
PMF.
3. Mass Spectrometry
Analysis of peptides using
mass spectrometry. The mass
spectrometric analysis
produces a list of molecular
weights of the fragments which
is often called a peak list.
4. In silico Digestion
In silico digestion of
database proteins and
generation of theoretical
peak list.
5. Comparison
Comparison of peak
list and theoretical
peak list to get best
match.
The Steps
….

6. SAMPLE PREPARATION
Chemical modification
Sample/ protease
50:1
Acetonitrile and vacuum
Distilled water and purified
MS

7. MALDI ANALYSIS
 A small fraction of the peptide (usually 1 microliter or less) is pipetted ontoa MALDI target and a
chemical called a matrix is added to the peptide mix
 The matrix molecules are required for the desorption of the peptide molecules.
 Matrix and peptide molecules co-crystallize on the MALDI target and are ready to be analyzed.
 The target is inserted into the vacuum chamber of the mass spectrometer and the desorption and
ionization of the polypeptide fragments is initiated by a pulsed laser beam which transfers high
amounts of energy into the matrix molecules.
 The energy transfer is sufficient to promote the ionization and transitionof matrix molecules and
peptides from the solid phase into the gas phase
 The ions are accelerated in the electric field of the mass spectrometer and fly towards an ion detector
where their arrival is detected as an electricsignal.
 Their mass-to-charge ratio is proportional to their time of flight (TOF) in the
drift tube and can be calculated accordingly.

9. list
Theabsolute masses of the peptides

10. Advantages
• Easy to perform
• No need for toomuch optimization
• It is significantly faster to carry out
than peptide sequencing
• Only the masses of the peptides need
to be known
• The protein sequence need to be
present in the database
• It often fails to identify mixture protein
• Peptides containing post-translational
modifications may not be correctly
identified
Disadvantages
….

11. Our PMF
service
MALDI/ESI TOF
analysis
Proteolytic digestion and
peptide extraction
Data report
Database search
Our services