i am sunita arora ph.d scholar in pantnagar university

1. Protein Epitope Mapping By Mass
Spectrometry
SUNITA
ID NO.55410
MBGE

2. INTRODUCTION
• the rapid mapping of linear epitopes in proteins that are
bound by mAb.
 Rapid determination of antigenic sites without tedious
peptide synthesis.
 coupling between matrix-assisted laser desorption mass
spectrometry and immunoprecipitation provides a
potentially powerful tool for determining binding sites
between proteins.

3. Matrix-assisted laser desorption mass
spectrometry (MALDI-MS)
measuring the molecular weights of peptides and
proteins.
• Accurate
• sensitive
• rapid identification of proteins

4. Rapid mapping of linear
protein epitopes
• Bound by monoclonal antibodies.
• Advantage of the purification power of
immunoprecipitation
•
• the high specificity and speed of MALDI-MS,

6. Requirement
• chymotrypsin,
• endoprotease Lys-C,
• serine protease inhibitor
• Protein G/protein A agarose
• Antimelittin monoclonal antibody (No. 83144)
• mouse IgGl
• Glucagon-like peptide-
• mouse monoclonal antibody

7. METHODS
• Consist three major step
• 1st step: an antigen protein is digested by a proteolytic
enzyme(produce an appropriate set of peptide
fragments).
• 2nd step: the linear epitope are selected and separated
from the pool of peptide fragments (by immuno
precipitation with the monoclonal antibody)
• 3rd step: peptides are identified (matrix assisted laser
desorption mass spectrometry)

8. How to perform
• Digestion of peptide:
• Protease and peptide was dissolved in an appropriate buffer
chymotrypsin and endoprotease lys-C.
The mixture is maintained at 37 0 C for 5-10 min we will get
partial digest of the peptide
mixed with protease and digested at 37 0C for 2 h, we will
obtain a complete digest of the peptide
• The digestions were terminated by addition of a one-tenth
volume Pefablock Sc solution by heating at 90 0C for 15 min.

9. Immunoprecipitation:
 mab (2-1 0 µg)and the mixture of peptides produced by the
proteolytic digestion mixed in n-octyl glucoside,(TSO)
 after a 2 h incubation at 4 0C with gentle stirring,
• 2-3 pL of protein G/protein A agarose was added to the
• solution and incubated for another 0.5 h at 4 OC.
• The agarose beads were collected by carefully the supernatant after
centrifugation of the solution for 1 min at 16000rpm.
• beads were washed three times. TSO buffer and mercaptoethanol,

10. • The laser desorption matrix and a suitable amount of internal standard
peptide(dynorphin A 1-13) were mixed with the washed beads.
• the bound peptides was loaded onto the mass spectrometer probe and
dried at room temperature with a stream of air for MALDI-MS analysis.
Mass Spectrometry.

12. Mass Spectrometry.
• MALDI-MS analysis was carried out on a laser desorption time-of-
flight instrument.
• entire triple complex consisting of proteinG/protein A agarose,
antibody,
• the bound peptide was subjected to mass spectrometric analysis.
• Intense peaks corresponding to the peptides that were bound to the
antibodies were observed
• the MALDI-MS conditions used in the present experiments cause
the peptide to dissociate from the antibody.
• dissociation may occur in the acidified matrix and/or during the
MALDI process.

13. • The mass spectra were collected by adding individual spectra
obtained from 200 laser shots to improve the statistics of the
measurement.
• calibrated using dynorphin A 1-1 3 and oxidized insulin B-chain.
• calibrations weremade using either the undigested parent peptide
and dynorphin A 1-13 or an external calibrant.

15. • Epitope Mapping of Melittin
• Matrix-assisted laser desorption mass spectra of peptide fragments
produced by endoprotease Lys-C digestion of melittin
• peptide fragments isolated by immuno precipitation with mab No.
83144.
• The peak labeled with an asterisk corresponds to the internally
added calibrant dynorphin A 1-13, and i1, and i2 refer to
unidentified impurities.

16. • Matrix assisted laser desorption mass spectra of peptide fragments
produced by chymotrypsin digestion of melittin
• Peptide fragments Isolated by immunoprecipitation with
antimelittin mab No. 83144.
• The peak labeled with an asterisk corresponds to the internally
added calibarnt dynorphin A 1-13,
• I refers to an unldentifted impurity.

17. • (a)Matrix-assisted laser desorption mass spectra of peptide
fragments produced by chymotrypsin digestion of GLP-17-37
• (b) peptide fragments isolated by immuno precipitation with
antiGLP-17-37 mab No. 26.1.
• The peak labeled with an asterisk corresponds to the internally
added calibrant dynorphin A 1-13,and I refers to an unidentified
Impurity.
• The satellite peaks labeled a, and a2 arise by adventitious Na and Cu
adduction to the peptide.22

18. • ( c). Amino acid sequence of GLP-17-37. The peptide fragments
produced by chymotrypsin digestion are shown by lines
• The solid lines represent those peptide fragments that were bound
by the antiGLP-1 7-37 mab No. 26.1
• the dashed lines those that were not bound by mab No. 26.1.

20. Epitope Mapping of GLP-1 7-37
• Matrix-assisted laser desorption mass spectra of
 peptide fragments produced by chymotrypsin digestion of GLP-1 7-37
 peptide fragments isolated by immunoprecipitation with antlGLP-17-37
mab No. 26.1.
• The peak labeled corresponds to the internally added calibrant dynorphin
• A 1-13, refers to an unidentified Impurity.
• The satellite peaks labeled a1 and a2 arise by Na and Cu adduction to the
peptide 22
• Amino acid sequence of GLP-17-37.
• The peptide fragments produced by chymotrypsin digestion are shown by
lines.
• The solid lines represent those peptide fragments thatiwere bound by the
antiGLP-1 7-37 mab No. 26.1
• dashed lines those that were not bound by mab No. 26.1.

22. Result
• Bee venom melittin and GLP- 1 7-37 were selected as modeL
Systems
• monoclonal antibodies were available that bind known linear
regions in these peptides.
• Antimelittin monoclonal antibody No. 83 144 was previously
determined to bind to an epitope located at residues 20-26 of
melittin,
• antiGLP- 1 7-37 monoclonal antibody No. 26.1 was previously
determined to bind a region that included the first three N-terminal
residues of GLP-17-37.

23. Conclusion
• Accurate determination of their molecular Weights
• by Selection and analysis of Antibody-binding peptides provide the
basis mass spectrometry
• Successfully demonstrated for an antibody
• low binding affinity (10-6)
• biomedical studies such as antigenic site mapping for polyclonal
antibodies
• determination of the sites of protein-protein
• or protein-ligand interaction

24. • Thank you