This document discusses peptide mapping, which involves enzymatically or chemically cleaving a protein into peptides and analyzing the peptides to identify the protein's primary structure. Peptide mapping is used to confirm identity, detect alterations, and ensure consistency for biopharmaceutical characterization. It involves four main steps - selectively cleaving peptide bonds, separating peptides chromatographically (often via RP-HPLC), analyzing and identifying peptides (usually via mass spectrometry), and comparing results to a reference standard to confirm identity. The document provides details on reagents, conditions, and parameters for each step of peptide mapping.

1. Guided by: Dr. Priti Mehta
Prepared by: Prachi Sacklecha
14mph305

2. Peptide mapping is a key part of the ICH Q6B guidelines for characterization and
confirmation of biopharmaceuticals in support of new marketing applications.
Note:
PEPTIDE MAPPING
 Peptide mapping is an identity test for proteins, especially those
obtained by r-DNA technology.
 Peptide mapping is a comparative procedure because the information
obtained, compared to a Reference Standard or Reference Material
similarly treated, confirms the primary structure of the protein, is
capable of detecting whether alterations in structure have occurred,
and demonstrates process consistency and genetic stability.
 Peptide mapping or PMF refers to the identification of proteins using
data from intact peptide masses.
 It is a powerful test that is capable of identifying single amino acid
changes resulting from events such as errors in the reading of
complementary DNA (cDNA) sequences or point mutations.

3. Principle
It involves the thermal chemical or enzymatic treatment of a
protein resulting in the formation of peptide fragments followed by
separation and identification of the fragments by mass spectroscopy
Peptide mapping is a comparative procedure because the information
obtained, compared to a Reference Standard or Reference Material
similarly treated
Digested peptide are analysed by MALDI-TOF MS in order to
determine the masses of intact peptide. These masses can be used in
correlative database searches to identified exact matches.

4. Applicatios:
To determined
location of amino
acid residues that
are post
trationationally
modified (Boyle
et al 1991) To prepared
individual
peptide to
determine A.
composition and
sequence.
N-terminal and
C-terminal
sequence
confirmation
It also used to
analysed proteins
that are not
radiolabeled.
It can also be
used to identified
epitopes
recognised by
specific Ab.
Disulfide bridge
assignment.

5. Four major steps
Note : Each step can be accomplished in different ways depending on
amount of protein available , the technologies availaible , and need of
researcher.
Analysis and identification of the peptides.
Chromatographic separation of the peptides.
Selective cleavage of the peptide bonds.
Isolation and purification of the protein.

6. Or HPLC

7. 95-100 % pure proteins are suitable for peptide mapping .
Isolation and Purification methods
Electrophoresis
• Gel elecrophoresis
• SDS-PAGE
• Iso electric focusing
• 2D-Electrophoresis
• Cappilary
electrophoresis
Chromatography
• Reverse-Phase High
Performance Liquid
• Chromatography (RP-
HPLC)
• Ion-Exchange
Chromatography (IEC)
• Hydrophobic Interaction
Chromatography (HIC)

8. SELECTIVE CLEAVAGE OF PEPTIDE BONDS
If two protein have same primary structure and then perform identical
cleavage reaction which yield identical peptide fragments. If protein
differ in structure , then cleavage yield dissimilar peptides. by comparing
banding pattern protein can be identify.
Two types of cleavage reagent
Enzymatic
• Advantages
• Easy to use
• Most reliable
• Safest
• Disadvantages
• They are themselves protein
so they interfere with result
• Membrane-spanning
segment-lack specific
proteolytic cleavage site
Chemical
• Advantages
• Easy to use
• Reliable
• Disadvantages
• Toxic
• Required careful handling.

9. Agent Specificity
Trypsin (EC 3.4.21.4) C-terminal side of Arg and Lys
Chymotrypsin (EC 3.4.21.1) C-terminal side of hydrophobic
residues(e.g., Leu, Met, Ala,)
Pepsin (EC 3.4.23.1 & 2) Nonspecific digest
Lysyl endopeptidase (Lys-C
Endopeptidase)
C-terminal side of Lys
Glutamyl endopeptidase (from S.
aureus strain V8)
C-terminal side of Glu andAsp
endopeptidase (Endoproteinase
Asp-N )
Peptidyl-Asp metallo
Clostripain (EC 3.4.22.8) N-terminal side of Asp
C-terminal side of Arg
Enzymes

10. Trypsin – catalyzes the hydrolytic cleavage of peptide bonds
Formed by the carboxyl groups of arginine and lysine.
N
H
O
H
N
O
N
H
R3
O
H
N
R1O
H3N
CO2
NH3
N
H
O
H
N
O
H3N
R3
O
H
N
R1O
H3N
CO2
NH3
O +
trypsin
H2O
means trypsin cleaves at the C-terminal side
of basic residues, Arg, Lys but not His
Chymotripsin - catalyzes the hydrolytic cleavage of peptide
bondsformed by the carboxyl groups of phenylalanine,
tyrosine, and tryptophan
means chymotrypsin: cleaves at the C-terminal side of aromatic
residues Phe, Tyr, Trp
N
H
O
H
N
O
N
H
R3
O
H
N
R1O
H3N
CO2 N
H
O
H
N
O
H3N
R3
O
H
N
R1O
H3N
CO2
O +
chymotrypsin
H2O

11. Agent Specificity
Cyanogen bromide C-terminal side of Met
2-Nitro-5-thio-cyanobenzoicAcid N-terminal side of Cys
O-iodosobenzoic acid C-terminal side of Trp and Tyr
Dilute acid Asp and Pro
CHEMICALS
Cyanogen bromide cleavage – specifically cleaves peptide
bonds formed from carboxyl group of methionine.

12. Pretreatment of cleavage agents—
• especially enzymatic agents—might be necessary for purification
purposes to ensure reproducibility of the map. For example, trypsin used
as a cleavage agent will have to be treated with tosyl-L-phenylalanine
chloromethyl ketone to inactivate chymotrypsin.
Pretreatment of the Protein
• Under certain conditions, it might be necessary to concentrate the sample
or to separate the protein from added substances and stabilizers used in
formulation of the product, if these interfere with the mapping procedure.
Physical procedures used for pretreatment can include ultrafiltration,
column chromatography, and lyophilization. .
• Other pretreatments, such as the addition of chaotropic agents (e.g., urea)
can be used to unfold the protein prior to mapping.

13. Conditions for optimal Digestion
pH
• experimentally
determined based on
the digestion agent.
• Cyanogen bromide:
acidic pH(pH2)
• Trypsin: pH 8
• pH should not affect the
protin or alter its
chemical integrity
should not affect the
fragmentation.
Temperature
• 25 -37°C for most
digestion.
• should not cause side
reaction/modification
in protine
• some proteins tend to
precipitate :based on
the type of protein.
Time
• determine using a
time course study
• Reaction should be
stopped by addition
of agents or freezing
to stop enzyme
activity.
Amount of
digesting agent:
• Protein to enzyme
ratio to be
determined • 20:1,
200:1 usually used
• Cleaving agent should
not be limiting – too
much excess will
interfere in the
chromatographic
pattern

14. CHROMATOGRAPHIC SEPARATION
 After cleavages , separation of peptide fragment
can be done by using several method.
 The selection of a technique depends on the
protein being mapped.
 A most widely used reverse-phase High
Performance Liquid Chromatographic (RP-HPLC)
method

15. RP-HPlC SYSTEM FOR PEPTIDE
MAPPING.
RP-Hplc instrumental condition for peptide mapping.
•Chromatographic Column—The selection of a chromatographic
column is empirically determined for each protein.
Columns with 100 Å or 300 Å pore size with silica support can give optimal
separation.
For smaller peptides
octylsilane chemically bonded to totally porous silica particles, 3 to 10 μm in
diameter (L7) .
octadecylsilane chemically bonded to porous silica or ceramic micro-particles,
3 to 10 μm in diameter (L1) column packings are more efficient than the butyl
silane chemically bonded to totally porous silica particles, 5 to 10 μm in diameter
(L26) packing.

16. 16
Solvent
The most commonly used solvent is water with acetonitrile as the
organic modifier to which less than 0.1% trifluoroacetic acid is
added.
If necessary, add isopropyl alcohol or n-propyl alcohol to
solubalize the digest components, provided that the addition does
not unduly increase the viscosity of the components.

17. Mobile Phase—
•Buffered mobile phases containing phosphate are used to provide some
flexibility in the selection of pH conditions, since shifts of pH in the 3.0 to 5.0
range enhance the separation of peptides containing acidic residues
(e.g.,glutamic and aspartic acids)
•Sodium or potassium phosphates, ammonium acetate, phosphoric acid, and a
pH between 2 and 7 have also been used with acetonitrile gradients.
• Acetonitrile containing trifluoroacetic acid is used quite often.
Gradient Selection—
• A shallow gradient is recommended in order to separate complex mixtures.
•Gradients are optimized to provide clear resolution of one or two peaks that
will become “marker” peaks for the test.

18. OTHERS
 TEMPERATURE CONTROL
-of the column is usually necessary to achieve good
reproducibility.
 THE FLOW RATES
range from 0.1 to 2.0 mL per minute.
 DETECTORS
-UV detector at 200 to 230 nm.
-Postcolumn derivatization (not as robust or
versatile as UV)
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19. System Suitability
This section provides an experimental means for
measuring the overall performance of the test method.
The acceptance criteria for system suitability depend on
the identification of critical test parameters that affect
data interpretation and acceptance.
An indicator that the desired digestion endpoint was
achieved is by the comparison with a Reference Standard,
which is treated exactly as the article under test.

20. The use of a Reference Standard or Reference Material in
parallel with the protein under test is critical in the
development and establishment of system suitability limits.
In addition a specimen chromatogram should be included with
the Reference Standard or Reference Material for additional
comparison purposes.
Other indicators may include visual inspection of protein or
peptide solubility, the absence of intact protein, or
measurement of responses of a digestion-dependent peptide.
The critical system suitability parameters for peptide analysis
will depend on the particular mode of peptide separation and
detection and on the data analysis requirements.

21. When peptide mapping is used as an identification test, the system
suitability requirements for the identified peptides covers selectivity
and precision.
The identification of the primary structure of the peptide fragments in
the peptide map provides both a verification of the known primary
structure and the identification of protein variants by comparison.
For an analysis of a variant protein, a characterized mixture of a variant
and a Reference Standard or Reference Material can be used, especially
if the variant peptide is located in a less-resolved region of the map.
Chromatographic parameters—such as peak-to-peak resolution,
maximum peak width, peak area, peak tailing factors, and column
efficiency—may be used to define peptide resolution.

22. The replicate analysis of the digest of the Reference Standard or Reference
Material for the protein under test yields measures of precision and
quantitative recovery.
Recovery of the identified peptides is generally ascertained by the use of
internal or external peptide standards.
Differences in the recovery and precision of the identified peptides are
expected; therefore, the system suitability limits will have to be established
for both the recovery and the precision of the identified peptides.
These limits are unique for a given protein and will be specified in the
individual monograph.

23. Visual comparison of the relative retention times, the peak responses, the
number of peaks, and the overall elution pattern is completed initially.
It is then complemented and supported by mathematical analysis of the
peak response ratios and by the chromatographic profile of a 1:1 (v/v)
mixture of sample and Reference Standard or Reference Material digest.
If all peaks in the sample digest and in the Reference Standard or
Reference Material digest have the same relative retention times and peaks
response ratios, then the identity of the sample under test is confirmed.
If peaks that initially eluted with significantly different relative retention
times are then observed as single peaks in the 1:1 mixture, the initial
difference would be an indication of system variability.

24. However, if separate peaks are observed in the 1:1 mixture, this would be
evidence of the nonequivalence of the peptides in each peak.
If a peak in the 1:1 mixture is significantly broader than the corresponding peak
in the sample and Reference Standard or Reference Material digest, it may
indicate the presence of different peptides.
Other automated approaches have been used that employ mathematical
formulas, models, and pattern recognition. Such approaches are, for example,
the automated identification of compounds by IR spectroscopy and the
application of diode-array UV spectral analysis for identification of peptides.
These methods have limitations due to inadequate resolutions, co-elution of
fragments, or absolute peak response differences between Reference Standard
or Reference Material and sample fragments.

25. ANALYSIS AND IDENTIFICATION OF
PEPTIDES
• Methods to characterize peaks range from N-
terminal sequencing of each peak followed by
amino acid analysis to the use of mass
spectroscopy (MS).
• Amino acid analysis of fragments may be
limited by the peptide size.
• If the N-terminus is blocked, it may need to be
• cleared before sequencing.
• C-terminal sequencing of proteins in
combination with carboxypeptidase and
MALDITOF-MS can also be used for
characterization purposes.
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