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PEPTIDE MAPPING

About Authors:
Gautambhai, Emanual Michael Patelia*, Arpit Shah
Department of Pharmaceutical Chemistry and Analysis,
Indukaka Ipcowala College of Pharmacy,
New Vallabh Vidyanagar – 388121, Gujarat, India.
*ricky.emanual@gmail.com

General Concept:

  • A fragmentation pattern generated by digestion of a particular protein with proteolytic enzymes of known specificity.
  • Used in protein identification.
  • Proteases will produce fragments of a characteristic size from a protein and this can be used as a test for the identity or otherwise of two similar sized proteins.
  • Peptide mapping involves controlled cleavage of a pure protein with small amounts of a pure protease to generate peptides of characteristic, reproducible sizes.

Introduction:

  • The comparison of the primary structure of proteins is an important facet in the characterization of families of proteins from the same organism, similar proteins from different organisms, and cloned gene products. A relatively uncomplicated approach is to compare the peptide fragments of proteins generated by enzymatic or chemical cleavage.
  • The principle behind peptide mapping is straightforward. If two proteins have the same primary structures, then cleavage of each protein with a specific protease or chemical cleavage reagent will yield identical peptide fragments.
  • However, if the proteins have different primary structures, then the cleavage will generate unrelated peptides. The similarity or dissimilarity of the protein's primary structure is reflected in the similarity or dissimilarity of the peptide fragments.
  • There are four phases of the peptide mapping process.
  1. Isolation and Purification of the protein, if the protein is part of a formulation.
  2. Radio labeling of the proteins, and thus the peptide fragments, to minimize the quantity of protein required.
  3. Cleavage of the proteins with specific endopeptidic reagents, either chemical or enzymatic.
  4. Separation and visualization of the peptide fragments for comparison.
  • 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.

The Peptide Map:

  • Peptide mapping is not a general method, but involves developing specific maps for each unique protein. Although the technology is evolving rapidly, there are certain methods that are generally accepted. Variations of these methods will be indicated, when appropriate, in specific monographs.
  • A peptide map may be viewed as a fingerprint of a protein and is the end product of several chemical processes that provide a comprehensive understanding of the protein being analyzed.
  • A test sample is digested and assayed in parallel with a Reference Standard or a Reference Material. Complete cleavage of peptide bonds is more likely to occur when enzymes such as endoproteases (e.g., trypsin) are used, instead of chemical cleavage reagents. A map should contain enough peptides to be meaningful. On the other hand, if there are too many fragments, the map might lose its specificity because many proteins will then have the same profiles.

Selective Cleavage of Peptide Bonds:

  • The selection of the approach used for the cleavage of peptide bonds will depend on the protein under test. This selection process involves determination of the type of cleavage to be employed either enzymatic or chemical, and the type of cleavage agent within the chosen category.

Type

Agent

Specificity

Enzymatic

Trypsin

C-terminal side of Arg and Lys


Chymotrypsin

C-terminal side of hydrophobic residues

(e.g., Leu, Met, Ala, aromatics)


Pepsin

Nonspecific digest


Glutamyl endopeptidase

C-terminal side of Glu and

Asp

Chemical

Cyanogen bromide

C-terminal side of Met


O-iodosobenzoic acid

C-terminal side of Trp and Tyr


Dilute acid

Asp and Pro


BNPS-skatole

Trp

Parameters to be looked after:
*    Pretreatment of Sample
*    Pretreatment of the Cleavage Agent
*    Pretreatment of the Protein
*    Establishment of Optimal Digestion Conditions
1.    pH
2.    Temperature
3.    Time
4.    Amount of Cleavage Agent

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Chromatographic Separation:

  • Many techniques are used to separate peptides for mapping. The selection of a technique depends on the protein being mapped.
  • Denatured proteins are digested to completion using a proteolytic enzyme and the peptides are resolved. The resulting patterns of bands, spots or peaks are referred to as peptide map.
  • Techniques that have been successfully used for separation of peptides are shown in table.

Techniques Used for the Separation of Peptides

Reverse-Phase High Performance Liquid Chromatography (RP-HPLC)

Ion-Exchange Chromatography (IEC)

Hydrophobic Interaction Chromatography (HIC)

Polyacrylamide Gel Electrophoresis (PAGE), nondenaturating

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Capillary Electrophoresis (CE)

Paper Chromatography-High Voltage (PCHV)

High-Voltage Paper Electrophoresis (HVPE)

Tryptic Mapping
Definition:

  • When trypsin is used as a protease for the digestion of proteins, the technique of peptide mapping is known as tryptic mapping.

Why trypsin?

  • High specificity (K or R, not followed by P)
  • Acetylated form commercially available (acetylation lessens auto digestion)
  • The possibility of auto-hydrolysis of trypsin is monitored by producing a blank peptide map, that is, the peptide map obtained when a blank solution is treated with trypsin.
  • Autolysis peaks are great internal calibrants.
  • Trypsin yields 47 peptides (theoretically)
  • In practice.......We see far fewer by mass spectrometry

Reasons
*       Possibly incomplete digestion

*       Lose of peptides during each manipulation
•      washes during digestion
•      washes during cleanup step
•      some peptides will not ionize well
•      some signals (peaks) are poor
•      low intensity; lack of resolution

Applications:

  • To compare the primary structure of proteins suspected of being encoded by the same or related genes.
  • To determine the precise location of amino acid residues that are post-translationally modified.
  • To locate the binding sites of antibodies and functionally important sites of the target protein.
  • To prepare individual peptides to determine amino acid composition and sequence.

Few practical examples:

  • IgG1 proteins
  • IgE receptor
  • Human chorionic gonadotropin
  • Recombinant bovine somatotropin
  • Recombinant human IL-2 and its variants
  • Recombinant DNA derived tissue plasminogen activator
  • Recombinant chimeric monoclonal antibody
  • Proteins which are especially obtained by rDNA technology

PEPTIDE MASS FINGERPRINTING

  • Peptide mapping by mass spectrometry (peptide mass fingerprinting) is used in protein characterization to produce a unique ‘fingerprint’ of an individual protein and to compare this with the theoretical gene-derived amino acid sequence. This analysis is used for identification purposes at all stages of drug discovery or to demonstrate comparability and consistency between batches for release during manufacturing. It may also be used for the characterization of reference batches.
  • It is a very powerful tool in protein characterization. Peptide mapping can also be used for:
  1. Dilsulfide bridge assignment
  2. N-terminal and C-terminal sequence confirmation (normally together with MS/MS sequencing)
  3. Screening and identification of sites of post-translational modification (e.g. glycosylation, phosphorylation)
  4. Guiding the choice of signals for subsequent MS/MS or gas phase sequence analysis
  • The use of MS for characterization of peptide fragments is by direct infusion of isolated peptides or for complex mixtures, on-line LC-MS can be used.
  • The protein molecule is fragmented using specific enzymatic or chemical methods and the resulting peptide mixture analyzed using MS, now the modern techniques of Electrospray (ES-MS) or MALDI TOF-MS.
  • Use of Fast Atom Bombardment MS (FAB-MS) is quite outdated now.
  • Tandem MS has also been used to sequence a modified protein and to determine the type of amino acid modification that has occurred.
  • The comparison of mass spectra of the digests before and after reduction provides a method to assign the disulfide bonds to the various sulfhydryl-containing peptides.
  • Peptide mapping is a key part of the ICH Q6B guidelines for characterization and confirmation of biopharmaceuticals in support of new marketing applications.

What you need for peptide mass mapping:
*       Some peptides

*       Protein Database
- GenBank, Swiss-Prot, dbEST, etc.

*       Search engines
- MasCot, Prospector, Sequest, etc.

Difference between peptide mapping and peptide sequencing:

Peptide mapping

Peptide sequencing

Protein must be in the database

Sequence information

Extremely sensitive

Sensitive

Clean sample

Extremely clean sample

Easy data acquisition

More complex data acquisition

Easy data interpretation

More complex data interpretation

Fast

Can be time consuming

Easy to automate

More difficult to automate

REFERENCES
*    nihs.go.jp/dbcb/Bio-Topic/peptide.pdf
*    Basic protein and peptide protocols By John M. Walker
*    Protein analysis and purification: benchtop techniques By Ian M. Rosenberg
*    academic.scranton.edu/organization/imbm/PMF_talk.ppt
*    m-scan.com/life-sciences-and-pharmaceutical/protein-peptide-analysis/peptide-mapping/

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