JANARDANA V & LAKSHMIPRIYA

SCHOOL OF LIFE SCIENCES CentralUniversity of TamilNadu

Protein Sequence Determination

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•Proteins central dogma

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proteins- amino acids( hair and skin) Amino Acids: Amine, Carboxylic Acid, R-group

carbohydrates- monosaccharide's, polysaccharides and disaccharides

Nucleic acids- nucleotides (make up the DNA)Nucleic Acids: Sugar, Phosphate, Base

lipids or fatty acids- glycerol

Proteins ♦ biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form in a biologically functional way.

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Protein structures

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There are 22 protein building amino acidsAmino Acids

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first described by the Dutch chemist Gerardus Johannes Mulder and named by the Swedish chemist Jöns Jacob Berzelius in 1838•1926, when James B. Sumner showed that the enzyme urease was in fact a protein

Pehr EdmanEdman reagent(phenylisothiocyanate (PITC))

Frederick sanger, 1953first protein to be sequenced was insulin.Sanger's reagent (1-fluoro-2,4-dinitrobenzene)

The first protein structures to be solved were Hemoglobin and Myoglobin, by Max Perutz and Sir John Cowdery Kendrew, 1958.X ray diffraction method

Methods protein sequencing1.Sub unit sequencing method2.Mass spectroscopy

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preliminary•Denature protein•Reduce and alkylate disulfides•Determine subunits

Analyze segments •Cleavage into segments•Edman degradation

Reconstruct sequences •Align sequences•Assign disulfides

Process of sequencing

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•unfolded from its compact globular shape into an extended linear form to make it more accessible to attack by chemical and enzymatic reagents.•e.g. → urea.

•2-mercaptoethanol(common reducing agent) or dithiothreitol (DTT) •disulfide bond is transferred from the polypeptide to the mercaptoethanols•In polypeptide sulfhydryl groups are formed & are treated with iodoacetic acid ( alkalising agent) →cysteine residues→ S—carboxymethylcysteine

•protein consists of only one type of polypeptide chain.•two different polypeptides can have the same N-terminal amino acid

01/05/2023

Reduce Disulfides

Denature Protein

Determine Subunits (Edman degradation )

♦ Polypeptide sequences longer than 40 to 100 residues cannot be directly sequenced

Certain enzymes cleave peptide bonds with great specificity e.g.→ Trypsin, will only hydrolyze peptide bonds after lysine or

arginine residues if the preceeding residue is not proline♦ In the case of ribonuclease A, trypsin digestion generates a total of

14 fragments. Chemical Fragmentation Methods:Cyanogen bromide (CNBr)

specifically cleaves Met residues at the C-end forming a homoserine lactone

CLEAVE INTO SEGMENTS

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N-terminal sequencingSANGER’S METHOD Treat with DNFB to form a derivative of the amino-terminal amino

acid hydrolysis Sanger's reagent (1-fluoro-2,4-dinitrobenzene Extraction of DNP-derivative with organic solvent Identification of DNP-derivative by chromatography and comparison

with standardsDANSYL CHLORIDE METHOD Forms a highly fluorescent derivative of the amino-terminal amino acid Identified by chromatography & fluorescence detection after acid

hydrolysis. Highly sensitive. Best for small amounts. EDMAN DEGRADATION Treat with phenylisothiocyanate(PITC) It removes one amino acid from the N-terminal end of the peptide under ideal conditions the sequence of 30-60 amino acids can be

determined

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♦ Edman degradation. phenylisothiocyanate o(PITC ) + N-terminal amino acid ----

→ PTC polypeptide.

1 2 stable PTH derivative

thiazolinone(derivative) 1 →PTC polypeptide is exposed to anhydrous trifluoroacetic acid2→ under acidic conditions, is converted to the more stable PTH

derivative, which can then be chromatographically identified

EDMAN DEGRADATION

• Acid catalyzed hydrolysis• ♦6M HCl/ 100-120°C/ 24 h (in oxygen free environment to

prevent oxidation of SH groups)

♦Some residues are degraded under these harsh conditions• Base catalyzed hydrolysis

- 4 M NaOH /100°C/ 4-8 hours-Arg, Cys, Ser and Thr are decomposed and other amino acids are deaminated and racemized.-Used mainly to determine Trp which is extensively degraded under acid catalyzed hydrolysis

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•EDMAN’S LIMITATIONS•The Edman degradation proceeds from the N-terminus of the protein it will not work it the N-terminal amino acid has been chemically modified.

•It also required the use of either guess work or a separate- procedure to determine the position of disulfide bridges.

• ADVANTAGES• The great advantage of the Edman method is that the rest of the peptide

chain after removal of the N-terminal amino acid is left intact for further cycles of this procedure, thus the Edman method can be used in a sequential fashion to identify several or many consecutive amino acid residues starting from the N-terminal end

Sanger’smethod

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Edman degradation

method

C-terminal sequencing• Add carboxypeptidases to a solution of the protein• Take samples at regular intervals• Determine the terminal amino acid by analyzing a plot of

amino acid concentrations against time.

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•N- and C- terminal Analysis-Exopeptidase Method

• Exopeptidases cleave the terminal residue of a polypeptide chain.

• Aminopeptidases cleave the N-terminal residues.

• Carboxypeptidases cleave the C-terminal residues.

• Further analyzed by amino acid analyzer.

HPLC APPATARUS

SOURCE: http://share.psu.ac.th/system/assets/media/files/000/000/674/original_HPLC.JPG?1306826838http://cbc.arizona.edu/classes/bioc462/462a/NOTES/Protein_Properties/Fig5_18cAffinityColumn.GIF

HPLC:HIGH PERFORMANCE LIQIUD CHROMATOGRAPHY

1. The amino acid derived from each cycle is identified by comparison to the retention time on reverse phase high performance liquid chromatography (HPLC/cLC) to the retention time of the PTH amino acid standards.

The mixture of fragments must be separated. An HPLC device will do this SEPARATION

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Compare amino acid sequence of one set of peptide fragments with the sequence of a second set of fragments obtained using different cleavage points.

Align sequences

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Automatic protein sequencers are designed that perform the 3 steps•labeling, separation and analysis of amino acids at a time and analyze data and give results automatically

AUTOMATED DEVICE IS DEVELPED BY EDMAN and GEOFFREY BEGG

Automatic protein sequencers

MASS SPECTROSCOPY METHOD1. This process is known as matrix assisted laser desorption /ionization mass

spectrometry(MALDI MS)2. Successfully used to measure the mass of a wide range of macromolecules.3. A solution of analyses is passed through a charged needle that is kept at a

high electrical potential, dispersing the solution into a fine charged micro droplets.

4. This create a spectrum of species with different mass to charge ratios.5. Each successive peak corresponds to a species that differs from that of its

neighboring peak by a charge difference of 1 and mass difference of 1(1 proton)

6. the mass of the protein can be determined from any two neighboring peaks.7. The measure of M/Z of one peak is • M is mass of the protein• N2 is the number of charges• X is the mass of the added groups

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Protein sequencing by Mass Spectrometry

•Mass spectrometry is an important emerging method for the characterization of proteins.

• The two primary methods for ionization of whole proteins are electro spray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI).

•Mass spectrometer has three main parts for characterizing the protein – 1. An ionization source 2. A mass analyzer 3. An ion detector

Working of Mass Spectrometry

Working of Mass Spectrometry

Working of Mass Spectrometry

Mass Spectrometry machine

Source: http://chemistry.umeche.maine.edu/CHY251/MassSpec1.https://www.uni-due.de/imperia/md/images/zmb/lcq-fleet-zmb-ace-ude.jpg

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ADVANTAGES –

•Stable isotopic labeling of proteins may be used to discriminate between contaminants and original partners using MS (mass spectrometry) techniques.

•MS (mass spectrometry) has also made advantages in the analysis of membrane proteins.

DIS-ADVANTAGES-

•Miscalibration is one of the main errors of MS spectrometry.

•MALDI doesn’t favor the identification of hydrophobic peptides.

• Determine number of polypeptide chains (subunits). • If subunits are too large, fragment them into shorter

polypeptide chains.• Determine number of disulfide bonds (inter-and intra-

chain).• Determine the amino acid composition of each

polypeptide chain.• Sequence each fragment using the Edman degradation

method.• Complete the sequence by comparing overlaps of

different sets of fragments.• For reference sequence , check it out in

BLAST,UNIPORT,NCBI.

protein

sequencing

method

SUMMARY

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Recombinant protein synthesisDrugs productionAntibiotic productionFunctional genomicsDetermine the protein folding patternsIn bioinformaticsIt plays vital role in proteomicsUsed for the prediction of final structure, function and location of proteinTo find out the location of gene coding for that protein

Applications of protein sequencing

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CLINICAL applications of protein sequencing

a) Identification of the protein family to which a particular protein belongs and finding the evolutionary history of that protein.

b) Prediction of the cellular localization of the protein based on its target sequence (sequence of amino acids at the N terminal end of the protein which determines the location of the protein inside the cell).

c) Prediction of the sequence of the gene encoding the particular protein.

d) Discovering the structure and function of a protein through various computational methods and experimental methods

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REFERNCES• Assignment of the disulfide bonds of huwentoxin-II by Edman degradation

sequencing and stepwise thiol modification.. Available from: https://www.researchgate.net/publication/12035017_Assignment_of_the_disulfidebonds_of_huwentoxinII_by_Edman_degradation_sequencing_and_stepwise_thiol_modification [accessed Oct 26, 2015].

• http://www.niaid.nih.gov/LabsAndResources/labs/aboutlabs/rtb/ProtChemSec/proteinSequencingUnit/technology/Pages/techEdman.aspx

• WIELY ONLINE LIBRARY http://higheredbcs.wiley.com/legacy/college/voet/0470129301/guided_exp/guided_exploration_4/protein_sequence.html

• Biochemistry- voet and voet• LEHNINGER’S, PRINCIPLES OF BIOCHEMISTRY.• WILSON &WALKER’S METHODS AND TECHNIQUES IN MOLECULAR

BIOLOGY AND BIOCHEMISTRY.• NCBI• BLAST• UNIPORT

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