peptide mapping

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Prepared by Masi hasmin h. (09mph411) PH:o8460857248 Peptide mapping NIRMA UNIVERSITY M.PHARM

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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. 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 . 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 or PMF refers to the identification of proteins using data from intact peptide masses.

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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 application To determined location of aminoacid residues that are posttrationationally modified (Boyle et al 1991) To prepared individual peptide to determine A.A composition and sequence. Dilsulfide bridge assignment It also used to analysed proteins that are not radiolabeled . It can also be used to identified epitopes recognised by specific Ab. N-terminal and C-terminal sequence confirmation 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.

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Four major steps isolation and purification of the protein, selective cleavage of the peptide bonds; chromatographic separation of the peptides analysis and identification of the peptides. Note : Each step can be accomplished in different ways depending on amount of protein available , the technologies availaible , and need of researcher.

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purification & Isolation or Chromatography 2-DE Mass Spectrometry MALDI-TOF MS -(LC)- ESI-MS/MS Clavages

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input into the computer program protein sequence databases for correlative searching.

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Database searching

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

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2-Dimension Electrophoresis ( 2-DE ) for Protein Separation The core technology of proteomics is 2-DE: At present, there is no other technique which is capable of resolving thousands of proteins in one separation procedure. Simultaneous separation and detection of ~2000 proteins on a 20x25 cm gel Up to 10,000 proteins can be seen using optimized protocols

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Blotting Protein sample 2 D-GE PVDF-membrane Blotting Digestion Blotting

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Detection

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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 Chemical Advantages Disadvantage Easy to use easy to use Most reliable Reliable Safest They are themselves protein so they interfere with result Toxic , required carefully handling Membrane-spanning segment-lack specific proteolytic cleavage site

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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 and Asp Peptidyl -Asp metallo endopeptidase ( Endoproteinase Asp-N ) N-terminal side of Asp Clostripain (EC 3.4.22.8) C-terminal side of Arg Enzymes Agent Specificity

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Trypsin – catalyzes the hydrolytic cleavage of peptide bonds Formed by the carboxyl groups of arginine and lysine. 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

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Which of these dipeptides is hydrolyzed by trypsin ? By chymotripsin ? . a. Arg - Glu -Ser . . b. Phe - Gly -Lys Problem : Lys C XXXXXK-- XXXXX Asp N endo XXXXXD-- XXXXX K-Lysine, R- Arginine , F-Phenylalanine, Y-Tyrosine, W- Tryptophan,D -Aspartic Acid, M- Methionine , P- Proline

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Problem : Which of these dipeptides is hydrolyzed by trypsin ? By chymotripsin ? a. Arg - Glu -Ser b. Phe - Gly -Lys trypsin Answer : Arg - Glu -Ser + H 2 O Arg + Glu -Ser chymotrypsin Phe - Gly -Lys + H 2 O Phe + Gly -Lys Trypsin and chymotrypsin are endopeptidases Carboxypeptidase : Cleaves the amide bond of the C-terminal amino acid ( exopeptidase ) NOTE:

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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 Agent Specificity Cyanogen bromide cleavage – specifically cleaves peptide bonds formed from carboxyl group of methionine . Chemicals CNBr XXXXXM--XXXXX

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Pretreatment of the Cleavage Agent 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.

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Establishment of Optimal Digestion Conditions Factors that affect the completeness and effectiveness of digestion of proteins are those that could affect any chemical or enzymatic reactions pH The pH of the digestion mixture is empirically determined to ensure the optimization of the performance of the given cleavage agent. example, when using cyanogen bromide as a cleavage agent, a highly acidic environment (e.g., pH 2, formic acid) is necessary; however, when using trypsin as a cleavage agent, a slightly alkaline environment (pH 8) is optimal. Temperature A temperature between 25EC and 37EC is adequate formost digestions. because some proteins are more susceptible to denaturation as the temperature of the reaction increases.

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Time Reaction time between protein and cleavage reagent is very important for optimum fragment and to avoid incomplete digestions Time of digestion varies from 2 to 30 hours. The reaction is stopped by the addition of an acid which does not interfere in the tryptic map or by freezing. Amount of Cleavage Agent A protein to protease ratio between 20:1 and 200:1 is generally used. It is recommended that the cleavage agent can be added in two or more stages to optimize cleavage. Although excessive amounts of cleavage agent are used to accomplish a reasonably rapid digestion time (i.e., 6 to 20 hours), the amount of cleavage agent is minimized to avoid its contribution to the chromatographic map pattern. Nonetheless, the final reaction volume remains small enough to facilitate the next step in peptide mapping—the separation step.

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Procedure Stain the PVDF membrane with 0.1 % Ponceau S and Identify the band of interest. Excise the band and cut the membrane into small pieces and place them in a 0.5ml microfuge tube. Add 90 mcl of appropriate Buffer and 10 mcl of chemical or enzymatic cleavage reagent Incubate with shaking at 37 C for 4 hr (for enzyme) or at room temperature for 24-48 hrs in dark under nitrogen (for chemical reagent) 0 Take supernant then evaporate to dryness on speedvac Then wash sample at least 4 time by adding 50 mcl of water then redrying again in speedvac . Now, sample is ready for peptide sepaation Note: chemical digestion will fail if protein is oxidized. So carryout under nitrogen

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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

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Methods for protein identification CIDspectra Est database

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If this approach fails , ESI-MS/MS analysis ca be used to generate peptide fragment ions. These can be used to produced de novo peptide sequences.

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After protein separation and fractionation, proteins are digested (their internal bonds are cut) by a specific enzyme. The result is a solution of many peptides derived from a single protein MALDI-TOF, can produce peptide mass fingerprints. The protein fingerprint is input into the program, and the program determines which sequence in the database would produce the same fingerprint as the one submitted. MASCOT www.matrixscience.com SWISS-PROT www.srs.ebi.ac.uk PepMAPPER http//wolf.bms.umist.ac.uk/ mapper Program

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ProFound http://129.85.19.192/profound_bin/WebProFound.exe MOWSE http://srs.hgmp.mrc.ac.uk/cgi-bin/mowse PeptideSearch http://www.narrador.embl-heidelberg.de/GroupPages/Homepage.html Mascot www.matrixscience.com PeptIdent http://us.expasy.org/tools/peptident.html OTHER WEB

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Protein Identification -Sequencing http://www.matrixscience.com

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Peptide Mapping Search Result

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REFERENCES Rosenberg Ian M. ,Protein analysis and purification Benchtop techniques, second Edition, Birkhauser Pp 211-253 Higgins D.,Taylor W., Bioinformatics sequence structure and databanks Practical apprpach ,5th indian edition ,oxford university ,Pp215-241 Heyer laurie J. , Discovery Genomic, proteomic ,and Bioinformatics,1 st Edition , New delhi,pearson education , Pp 184-187 Twyman R. M. , Principles of Proteomics , New York , Taylor and francies publisher , Pp55-67 USP www.sciencedirect.com

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