PROTEIN SEQUENCING. First Sequence

Save this PDF as:
 WORD  PNG  TXT  JPG

Size: px
Start display at page:

Download "PROTEIN SEQUENCING. First Sequence"

Transcription

1 PROTEIN SEQUENCING First Sequence The first protein sequencing was achieved by Frederic Sanger in He determined the amino acid sequence of bovine insulin Sanger was awarded the Nobel Prize in

2 I. Strategy Determine number of polypeptide chains (subunits) Determine number of disulfide bonds (inter- and intrachain) Determine the amino acid composition of each polypeptide chain If subunits are too large, fragment them into shorter polypeptide chains Sequence each fragment using the Edman degradation method Complete the sequence by comparing overlaps of different sets of fragments II. End-group Analysis Number of chains can be determine by identifying the number of N- and C-terminal. N-terminal analysis Dansyl chloride Phenylisothiocynate (PITC)/ Edman reagent Aminopeptidase C-terminal analysis carboxypeptidase 2

3 N-terminal Analysis with Dansyl Chloride Main reagent: 1-dimethyl aminophthalene-5-sulfonyl chloride (dansyl chloride) Dansyl poplypeptide chain is prepared Acidic hydrolysis liberates all amino acid and the N- terminal dansyl amino acid Amino acids are separated Fluorescence of the dansyl amino acid is detected Type of aa is obtained from comparison with standard dansylated amino acids N-terminal Analysis Edman (Degradation) Nucleophilic attack on phenyl isothiocyanate (PITC), the Edman reagent, under mild alkaline conditions (Nmethylpiperidine/water/ methanol) Formation of a phenylthiocarbamyl derivative (PTC-peptide) 3

4 N-terminal Analysis Edman (Degradation) Anhydrous trifluoro acetic acid (TFA) is used to cleave the terminal amino acid in the form of a thiozolinone derivative leaving the other peptide bonds intact The thiozolinone (TZ) derivative is extracted in an organic solvent (e.g. N-butyl chloride) Peptide cleaved carries a free amino terminus N-terminal Analysis Edman (Degradation) The TZ is extracted into an organic solvent and treated with an acid (25 % TFA/water) to form phenylthiohydantoin (PTH) derivative PTH is detected from UV absorption at 296 nm 4

5 N-terminal Analysis-Edman Degradation PTH amino acid is separated from the other components by chromatography or electrophoresis The terminal amino is identified according to retention time or mass This sequence can be repeated to identify all amino acid in short peptide chains (40-60 amino acid long) Edman Degradation on Protein Sequencer Perkin Elmer Applied Biosystems Model 494 Procise protein/peptide sequencer 5

6 Edman Degradation on Protein Sequencer By-products of Edman Degradation 6

7 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 Aminopeptidases and carboxypeptidases are highly specific, thus are of limited use due to slow rates and resistance of some amino to cleavage III. Disulfide Bond Cleavage Disulfides are reduced to thiol with dithiothreitol (DTT) or 2- mercaptoethanol Thiols are treated with alkylating agents (e.g. iodoacetic acid) to prevent the re-oxidation during subsequent steps. 7

8 Protection of sulfyhydryl groups IV. Separation and Molecular Weight Determination of Subunits Traditional Methods SDS-PAGE, SEC, or RP-HPLC are used to separate the subunits after cleavage of disulfide bonds Mw standards and a calibration curve are used to determine the molecular weights The approximate number of amino acids can be estimated from the Mw of the subunit using 110 Da as the average molar mass for each amino acid Recent methods MALDI: more accurate and fast 8

9 V. Amino Acid composition Strategy: hydrolysis followed by separation and identification Acid catalyzed hydrolysis 6M HCl/ ºC/ 24 h (in oxygen free environment to prevent oxidation of SH groups) Some residues are degrated under these harsh conditions Base catalyzed hydrolysis 4 M NaOH /100ºC/ 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 V. Amino Acid composition Enzymatic hydrolysis By exo- and endopeptidases A combination of endo and exopeptidases must be used to hydrolyze all the peptide bonds Separation Individual amino acids in hydrolyzed mixture can be separated by RP-HPLC or CE and identified according to retention time Increasing sensitivity Pre- or post-column derivatization is used to increase sensitivity 9

10 Derivatization with OPA and MCE VI. Cleavage of Specific Peptide Bonds Direct sequencing is applicable to peptides that have up to about 50 residues only. Problems which occur after lengthy reactions Incomplete reactions Accumulation of impurities from side reactions Solution: use enzymes to break down the polypeptide chain into shorter fragments Proteolytic enzymes: endopeptidases and exopeptidases 10

11 Enzymatic Fragmentation Trypsin Trypsin is the most commonly used proteolytic enzyme. It cleaves at the C-end of positively charged amino acids (Arg and Lys) if the next residue is not a proline. It is highly specific Cleavage sites may be removed or added via derivatization to take advantage of the specificity of trypsin Reaction times can be adjusted to limit proteolysis if there are too many Arg and Lys residues Non-denaturing conditions can be used to limit proteolysis as well Trypsin Digestion 11

12 Derivatization of Cys for Tryptic Digestion Other Proteolytic Enzymes Endopeptidases Pepsin; cleaves at the amino end of Phe, Tyr, Trp the previous residue is not a proline Chymotrypsin: cleaves at the carboxyl end of Phe, Trp, Tyr if the next residue is not proline Endopeptidae GluC: cleaves at the carboxy end of Glu Exopeptidases Leucine aminopeptidase: cleaves rapidly N-terminal leucine aa. Does not cleave N-terminal proline Aminopeptidase M: cleaves all N-terminal residues Carboxypeptidase A: cleaves all except Arg, Lys, and Pro Especially efficient for aa with bulky aliphatic and aromatic side chains Does not cleave if the next residue is Pro Carboxypeptidase B: cleaves C-terminal Arg and Lys if the next residue is not Pro Carboxypeptidase C: cleaves C-terminal residues 12

13 Chemical Fragmentation Methods Cyanogen bromide (CNBr) specifically cleaves Met residues at the C-end forming a homoserine lactone Sequence Determination Separate segments by chromatography or electrophoresis and sequence fragments individually Edman degradation is the method of choice Fully automated systems which use the Edman degradation methods are available commercially (Sequenator) In the sequenator the protein is immobilized through bonding to a solid support or by adsorbing it onto an inert glass frit. Controlled amounts of reagents are injected by a pumping system The thiozolinone is transferred to a conversion chamber for hydrolysis to the PTH amino acid The final product, the PTH amino acid, is pumped into an HPLC column for on-line analysis 1 hour analysis time is possible for 50 amino acid residues 13

14 The solid-phase matrix-the Merrifield resin Edman degradation 14

15 Ordering of Peptide Fragments Compare amino acid sequence of one set of peptide fragments with the sequence of a second set of fragments obtained using different cleavage points Determination of Disulfide Bond Position Digest polypeptide chain(s) Run 2D gel of mixture of fragments using same conditions in both dimension After separation in the first dimension, the matrix is exposed to performic acid which cleaves all possible disulfide bonds Separation in the second dimension is performed Fragment without ss bonds will be positioned along the diagonal of the matrix Fragments linked by S-S bonds will produce off diagonal spots The disulfide linked fragments can be extracted from the gel and sequenced 15

16 Protein Sequencing by Mass Spectrometry Digest protein Obtain MALD TOF mass spectrum of digest Use online database to match fragments patterns with those in the data base Obtain sequence of fragments by performing MS/MS 16

Lecture 18: Protein Sequencing

Lecture 18: Protein Sequencing Lecture 18: Protein Sequencing Frederic Sanger first time achieved complete sequence of protein (bovine insulin) in 1953. For his work, he was awarded the Nobel Prize of Chemistry in (1958). Protein sequencing

More information

--not necessarily a protein! (all proteins are polypeptides, but the converse is not true)

--not necessarily a protein! (all proteins are polypeptides, but the converse is not true) 00Note Set 5b 1 PEPTIDE BONDS AND POLYPEPTIDES OLIGOPEPTIDE: --chain containing only a few amino acids (see tetrapaptide, Fig 5.9) POLYPEPTIDE CHAINS: --many amino acids joined together --not necessarily

More information

Biochemistry - I. Prof. S. Dasgupta Department of Chemistry Indian Institute of Technology, Kharagpur Lecture-11 Enzyme Mechanisms II

Biochemistry - I. Prof. S. Dasgupta Department of Chemistry Indian Institute of Technology, Kharagpur Lecture-11 Enzyme Mechanisms II Biochemistry - I Prof. S. Dasgupta Department of Chemistry Indian Institute of Technology, Kharagpur Lecture-11 Enzyme Mechanisms II In the last class we studied the enzyme mechanisms of ribonuclease A

More information

Covalent bonds are the strongest chemical bonds contributing to the protein structure A peptide bond is formed between with of the following?

Covalent bonds are the strongest chemical bonds contributing to the protein structure A peptide bond is formed between with of the following? MCAT Question Covalent bonds are the strongest chemical bonds contributing to the protein structure A peptide bond is formed between with of the following? A. Carboxylic group and amino group B. Two carboxylic

More information

Marmara Üniversitesi Fen-Edebiyat Fakültesi Kimya Bölümü / Biyokimya Anabilim Dalı

Marmara Üniversitesi Fen-Edebiyat Fakültesi Kimya Bölümü / Biyokimya Anabilim Dalı EXPERIMENT IX Marmara Üniversitesi DETERMINATION OF N-TERMINAL AMINO ACID RESIDUE OF PROTEINS BY THIN LAYER CHROMATOGRAPHY Functions of the proteins depend upon its amino acid sequence. Because amino acid

More information

A. A peptide with 12 amino acids has the following amino acid composition: 2 Met, 1 Tyr, 1 Trp, 2 Glu, 1 Lys, 1 Arg, 1 Thr, 1 Asn, 1 Ile, 1 Cys

A. A peptide with 12 amino acids has the following amino acid composition: 2 Met, 1 Tyr, 1 Trp, 2 Glu, 1 Lys, 1 Arg, 1 Thr, 1 Asn, 1 Ile, 1 Cys Questions- Proteins & Enzymes A. A peptide with 12 amino acids has the following amino acid composition: 2 Met, 1 Tyr, 1 Trp, 2 Glu, 1 Lys, 1 Arg, 1 Thr, 1 Asn, 1 Ile, 1 Cys Reaction of the intact peptide

More information

(c) How would your answers to problem (a) change if the molecular weight of the protein was 100,000 Dalton?

(c) How would your answers to problem (a) change if the molecular weight of the protein was 100,000 Dalton? Problem 1. (12 points total, 4 points each) The molecular weight of an unspecified protein, at physiological conditions, is 70,000 Dalton, as determined by sedimentation equilibrium measurements and by

More information

Lecture 13-14 Conformation of proteins Conformation of a protein three-dimensional structure native state. native condition

Lecture 13-14 Conformation of proteins Conformation of a protein  three-dimensional structure native state. native condition Lecture 13-14 Conformation of proteins Conformation of a protein refers to the three-dimensional structure in its native state. There are many different possible conformations for a molecule as large as

More information

The Organic Chemistry of Amino Acids, Peptides, and Proteins

The Organic Chemistry of Amino Acids, Peptides, and Proteins Essential rganic Chemistry Chapter 16 The rganic Chemistry of Amino Acids, Peptides, and Proteins Amino Acids a-amino carboxylic acids. The building blocks from which proteins are made. H 2 N C 2 H Note:

More information

Amino Acids, Peptides and Proteins

Amino Acids, Peptides and Proteins Amino Acids, Peptides and Proteins 1. α-amino Acids C (S) or L amino acids a) dipolar nature (isoelectric points) b) synthesis (racemic) i) from α-bromoacids ii) Strecker synthesis from aldehydes iii)

More information

Methods for Protein Analysis

Methods for Protein Analysis Methods for Protein Analysis 1. Protein Separation Methods The following is a quick review of some common methods used for protein separation: SDS-PAGE (SDS-polyacrylamide gel electrophoresis) separates

More information

Ionization of amino acids

Ionization of amino acids Amino Acids 20 common amino acids there are others found naturally but much less frequently Common structure for amino acid COOH, -NH 2, H and R functional groups all attached to the a carbon Ionization

More information

The chemistry of insulin

The chemistry of insulin FREDERICK S ANGER The chemistry of insulin Nobel Lecture, December 11, 1958 It is great pleasure and privilege for me to give an account of my work on protein structure and I am deeply sensitive of the

More information

Food Proteins. Prof. Dr. Mohamed Fawzy Ramadan Hassanien Zagazig University, Egypt

Food Proteins. Prof. Dr. Mohamed Fawzy Ramadan Hassanien Zagazig University, Egypt Food Proteins Prof. Dr. Mohamed Fawzy Ramadan Hassanien Zagazig University, Egypt -Amino Acid Sequence -Protein Conformation -Levels of Protein Structure -Primary structure -Secondary structure -Tertiary

More information

Chapter 26 Biomolecules: Amino Acids, Peptides, and Proteins

Chapter 26 Biomolecules: Amino Acids, Peptides, and Proteins John E. McMurry www.cengage.com/chemistry/mcmurry Chapter 26 Biomolecules: Amino Acids, Peptides, and Proteins Proteins Amides from Amino Acids Amino acids contain a basic amino group and an acidic carboxyl

More information

H H N - C - C 2 R. Three possible forms (not counting R group) depending on ph

H H N - C - C 2 R. Three possible forms (not counting R group) depending on ph Amino acids - 0 common amino acids there are others found naturally but much less frequently - Common structure for amino acid - C, -N, and functional groups all attached to the alpha carbon N - C - C

More information

GSAK YLDR WGSM. (b) (5 pts) Explain why neither of these steps alone is sufficient to unambiguously determine the sequence of your peptide.

GSAK YLDR WGSM. (b) (5 pts) Explain why neither of these steps alone is sufficient to unambiguously determine the sequence of your peptide. Problem 1. (total 30 points) You have to determine the amino acid sequence of a peptide. You performed the following steps using enzyme cleavage of your peptide (see table on the front page) combined with

More information

IV. -Amino Acids: carboxyl and amino groups bonded to -Carbon. V. Polypeptides and Proteins

IV. -Amino Acids: carboxyl and amino groups bonded to -Carbon. V. Polypeptides and Proteins IV. -Amino Acids: carboxyl and amino groups bonded to -Carbon A. Acid/Base properties 1. carboxyl group is proton donor! weak acid 2. amino group is proton acceptor! weak base 3. At physiological ph: H

More information

Mass spectrometry based proteomics

Mass spectrometry based proteomics Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities TÁMOP-4.1.1.C-13/1/KONV-2014-0001 Mass

More information

Peptide bonds: resonance structure. Properties of proteins: Peptide bonds and side chains. Dihedral angles. Peptide bond. Protein physics, Lecture 5

Peptide bonds: resonance structure. Properties of proteins: Peptide bonds and side chains. Dihedral angles. Peptide bond. Protein physics, Lecture 5 Protein physics, Lecture 5 Peptide bonds: resonance structure Properties of proteins: Peptide bonds and side chains Proteins are linear polymers However, the peptide binds and side chains restrict conformational

More information

General Protein Metabolism

General Protein Metabolism General Protein Metabolism Protein Digestion Dietary proteins are very large complex molecules that cannot be absorbed from the intestine. To be absorbed, dietary proteins must be digested to small simple

More information

Peptides: Synthesis and Biological Interest

Peptides: Synthesis and Biological Interest Peptides: Synthesis and Biological Interest Therapeutic Agents Therapeutic peptides approved by the FDA (2009-2011) 3 Proteins Biopolymers of α-amino acids. Amino acids are joined by peptide bond. They

More information

Amino Acids, Peptides, Proteins

Amino Acids, Peptides, Proteins Amino Acids, Peptides, Proteins Functions of proteins: Enzymes Transport and Storage Motion, muscle contraction Hormones Mechanical support Immune protection (Antibodies) Generate and transmit nerve impulses

More information

Lecture 4: Peptides and Protein Primary Structure [PDF] Key Concepts. Objectives See also posted Peptide/pH/Ionization practice problems.

Lecture 4: Peptides and Protein Primary Structure [PDF] Key Concepts. Objectives See also posted Peptide/pH/Ionization practice problems. Lecture 4: Peptides and Protein Primary Structure [PDF] Reading: Berg, Tymoczko & Stryer, Chapter 2, pp. 34-37 Practice problems (peptide ionization) [PDF]; problems in textbook: chapter 2, pp. 63-64,

More information

2. Couple the two protected amino acids.

2. Couple the two protected amino acids. General Considerations The Strategy of Peptide Synthesis Making peptide bonds between amino acids is not difficult. The challenge is connecting amino acids in the correct sequence. andom peptide bond formation

More information

Chapter 27: Amino Acids, Peptides, and Proteins. monomer unit: α-amino acids

Chapter 27: Amino Acids, Peptides, and Proteins. monomer unit: α-amino acids Chapter 27: Amino Acids, Peptides, and Proteins. monomer unit: αamino acids 2 C 2! Amino Acid = sidechain Biopolymer: the monomeric amino acids are linked through an amide bond (the carboxylic acids of

More information

Guidance for Industry

Guidance for Industry Guidance for Industry for the Submission of Chemistry, Manufacturing, and Controls Information for Synthetic Peptide Substances Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation

More information

USP's Therapeutic Peptides Expert Panel discusses manufacturing processes and impurity control for synthetic peptide APIs.

USP's Therapeutic Peptides Expert Panel discusses manufacturing processes and impurity control for synthetic peptide APIs. Control Strategies for Synthetic Therapeutic Peptide APIs Part III: Manufacturing Process Considerations By Brian Gregg,Aleksander Swietlow,Anita Y. Szajek,Harold Rode,Michael Verlander,Ivo Eggen USP's

More information

Proteins are polymers of amino acids. Protein-over 50 amino acids, peptide-under 50 amino acids.

Proteins are polymers of amino acids. Protein-over 50 amino acids, peptide-under 50 amino acids. Amino Acids and Proteins: Protein Functions: enzymes, transport (hemoglobin-o 2, tranferrin-fe), protection (MHC molecules, immunoglobulins), hormones (insulin, glucagons), gene transcription regulation

More information

Recap. Lecture 2. Protein conformation. Proteins. 8 types of protein function 10/21/10. Proteins.. > 50% dry weight of a cell

Recap. Lecture 2. Protein conformation. Proteins. 8 types of protein function 10/21/10. Proteins.. > 50% dry weight of a cell Lecture 2 Protein conformation ecap Proteins.. > 50% dry weight of a cell ell s building blocks and molecular tools. More important than genes A large variety of functions http://www.tcd.ie/biochemistry/courses/jf_lectures.php

More information

Protein Physics. A. V. Finkelstein & O. B. Ptitsyn LECTURE 1

Protein Physics. A. V. Finkelstein & O. B. Ptitsyn LECTURE 1 Protein Physics A. V. Finkelstein & O. B. Ptitsyn LECTURE 1 PROTEINS Functions in a Cell MOLECULAR MACHINES BUILDING BLOCKS of a CELL ARMS of a CELL ENZYMES - enzymatic catalysis of biochemical reactions

More information

Advanced Medicinal & Pharmaceutical Chemistry CHEM 5412 Dept. of Chemistry, TAMUK

Advanced Medicinal & Pharmaceutical Chemistry CHEM 5412 Dept. of Chemistry, TAMUK Advanced Medicinal & Pharmaceutical Chemistry CHEM 5412 Dept. of Chemistry, TAMUK Dai Lu, Ph.D. dlu@tamhsc.edu Tel: 361-221-0745 Office: RCOP, Room 307 Drug Discovery and Development Drug Molecules Medicinal

More information

Dr. Rita P.-Y. Chen Institute of Biological Chemistry Academia Sinica

Dr. Rita P.-Y. Chen Institute of Biological Chemistry Academia Sinica PEPTIDE SYNTHESIS Dr. Rita P.-Y. Chen Institute of Biological Chemistry Academia Sinica 1 Solution phase chemistry -Time consuming: isolation and purification at each step -Low yield: can t drive reaction

More information

PRACTICAL 3: DIGESTIVE ENZYMES, SPECIFICITY AND ph

PRACTICAL 3: DIGESTIVE ENZYMES, SPECIFICITY AND ph PRACTICAL 3: DIGESTIVE ENZYMES, SPECIFICITY AND ph 3.1 Introduction The aims of this practical are: to illustrate the different ph dependence of gastric and pancreatic digestive proteases to illustrate

More information

Introduction to Chemical Biology

Introduction to Chemical Biology Professor Stuart Conway Introduction to Chemical Biology University of xford Introduction to Chemical Biology ecommended books: Professor Stuart Conway Department of Chemistry, Chemistry esearch Laboratory,

More information

Protein Purification and Analysis

Protein Purification and Analysis Protein Purification and Analysis Numbers of genes: Humans ~40,000 genes Yeast ~6000 genes Bacteria ~3000 genes Solubility of proteins important for purification: 60-80% soluble, 20-40% membrane Some proteins

More information

Biochemistry 2000 Sample Question Proteins. (1) Identify the secondary structure described in each of the following statements:

Biochemistry 2000 Sample Question Proteins. (1) Identify the secondary structure described in each of the following statements: (1) Identify the secondary structure described in each of the following statements: a. A coiled peptide chain held in place by hydrogen bonding between peptide bonds in the same chain b. A structure that

More information

Part A: Amino Acids and Peptides (Is the peptide IAG the same as the peptide GAI?)

Part A: Amino Acids and Peptides (Is the peptide IAG the same as the peptide GAI?) ChemActivity 46 Amino Acids, Polypeptides and Proteins 1 ChemActivity 46 Part A: Amino Acids and Peptides (Is the peptide IAG the same as the peptide GAI?) Model 1: The 20 Amino Acids at Biological p See

More information

1) Technical informations. - a) How does it work? - b) Purification - c) Quality Control. 2) Standard synthesis

1) Technical informations. - a) How does it work? - b) Purification - c) Quality Control. 2) Standard synthesis 1) Technical informations - a) How does it work? - b) Purification - c) Quality Control 2) Standard synthesis - a) Standard peptides - b) Modified peptides - c) Shipment and Delivery Time - d) How to order?

More information

Ch7 Enzymes II: Coenzymes, Regulation, Abzymes, and Ribozymes 阮雪芬 NTU

Ch7 Enzymes II: Coenzymes, Regulation, Abzymes, and Ribozymes 阮雪芬 NTU Ch7 Enzymes II: Coenzymes, Regulation, Abzymes, and Ribozymes 阮雪芬 2004/04/23 @ NTU Enzyme: Coenzyme Partners Vitamins and coenzymes Coenzyme: an organic or organometallic molecule that assists an enzyme.

More information

Outline. Market & Technology Trends. LifeTein Technology Portfolio. LifeTein Services

Outline. Market & Technology Trends. LifeTein Technology Portfolio. LifeTein Services 1 Outline Market & Technology Trends LifeTein Technology Portfolio LifeTein Services 2 Synthetic Therapeutic Peptides More than 60 synthetic therapeutic peptides under 50 amino acids in size have reached

More information

Definition of the Measurand: CRP

Definition of the Measurand: CRP A Reference Measurement System for C-reactive Protein David M. Bunk, Ph.D. Chemical Science and Technology Laboratory National Institute of Standards and Technology Definition of the Measurand: Human C-reactive

More information

Protein quantification and detection methods

Protein quantification and detection methods Protein quantification and detection methods 1) Spectroscopic procedures 2) Measurement of the total protein content by colorimetry 3) Amino acid analysis 4) Other methods, eg. radiolabelling of proteins,

More information

Pipe Cleaner Proteins. Essential question: How does the structure of proteins relate to their function in the cell?

Pipe Cleaner Proteins. Essential question: How does the structure of proteins relate to their function in the cell? Pipe Cleaner Proteins GPS: SB1 Students will analyze the nature of the relationships between structures and functions in living cells. Essential question: How does the structure of proteins relate to their

More information

An In-Gel Digestion Protocol

An In-Gel Digestion Protocol An In-Gel Digestion Protocol This protocol describes the digestion of a protein present in an SDS-PAGE gel band with trypsin. The band can be taken from either a 1D or 2D electrophoresis gel. Reagents

More information

Amino Acid Analysis of Pure Protein Hydrolysate with Waters UPLC Amino Acid Analysis Solution

Amino Acid Analysis of Pure Protein Hydrolysate with Waters UPLC Amino Acid Analysis Solution Amino Acid Analysis of Pure Protein Hydrolysate with Waters UPLC Amino Acid Analysis Solution Hillary B. Hewitson, Thomas E. Wheat, and Diane M. Diehl INTRODUCTION Amino acid analysis is used in the protein

More information

ORIGINAL SCIENTIFIC PAPER. Gabriella POHN. Éva VARGA-VISI SUMMARY KEY WORDS

ORIGINAL SCIENTIFIC PAPER. Gabriella POHN. Éva VARGA-VISI SUMMARY KEY WORDS ORIGINAL SCIENTIFIC PAPER 269 Determination of the Enantiomers of Methionine and Cyst(e)ine in the Form of Methionine-sulphon and Cysteic Acid After Performic Acid Oxidation by Reversed Phase High Performance

More information

A Guide to the Analysis and Purification of Proteins and Peptides by Reversed-Phase HPLC

A Guide to the Analysis and Purification of Proteins and Peptides by Reversed-Phase HPLC A Guide to the Analysis and Purification of Proteins and Peptides by Reversed-Phase HPLC HPLC Columns David Carr Your decision has lasting effects. Choose wisely. HPLC Columns Ultra Inert Base-Deactivated

More information

SpikeTides TM Peptides for relative and absolute quantification in SRM and MRM Assays

SpikeTides TM Peptides for relative and absolute quantification in SRM and MRM Assays Protocol SpikeTides TM Peptides for relative and absolute quantification in SRM and MRM Assays Contact us: InfoLine: +49-30-6392-7878 Order per fax: +49-30-6392-7888 or e-mail: www: peptide@jpt.com www.jpt.com

More information

membrane was isolated from H. halobium and apomembrane

membrane was isolated from H. halobium and apomembrane Proc. Natl. Acad. Sci. USA Vol. 76, No. 1, pp. 546-55, October 1979 Biochemistry Amino acid sequence of bacteriorhodopsin (purple membrane/hydrophobic peptides/high-pressure liquid chromatography/gas chromatographic

More information

It s the amino acids!

It s the amino acids! Catalytic Mechanisms HOW do enzymes do their job? Reducing activation energy sure, but HOW does an enzyme catalysis reduce the energy barrier ΔG? Remember: The rate of a chemical reaction of substrate

More information

Chapter 19 Amino Acids and Proteins

Chapter 19 Amino Acids and Proteins Chapter 19 Amino Acids and Proteins 19.1 Proteins and Amino Acids 19.2 Amino Acids as Acids and Bases Copyright 2007 by Pearson Education, Inc. Publishing as Benjamin Cummings 1 Functions of Proteins Proteins

More information

Supporting Information. Minimum active structure of insulin-like. peptide 5 (INSL5)

Supporting Information. Minimum active structure of insulin-like. peptide 5 (INSL5) Supporting Information Minimum active structure of insulin-like peptide 5 (INSL5) Alessia Belgi 1,2, Ross A.D. Bathgate *1,2,3, Martina Kocan *4, Nitin Patil 1,5, Suode Zhang 1, Geoffrey W. Tregear 1,2,

More information

The amino acids differ in the properties of their side chains. Hydrophobic, non acidic (the H+ ion won t associate with water)

The amino acids differ in the properties of their side chains. Hydrophobic, non acidic (the H+ ion won t associate with water) Amino Acids 101 What is an amino acid? Amino acids, or alpha- amino acids, are the building blocks of peptides and proteins They are composed of amine and carboxylic acid groups, separated by the alpha-carbon

More information

Application Note. Determination of Amino acids by UHPLC with automated OPA- Derivatization by the Autosampler. Summary. Fig. 1.

Application Note. Determination of Amino acids by UHPLC with automated OPA- Derivatization by the Autosampler. Summary. Fig. 1. Application Note Determination of Amino acids by UHPLC with automated PA- Derivatization by the Autosampler Category Bio Analysis Matrix - Method UHPLC Keywords Proteinogenic Amino acids, Canonical Amino

More information

Chem 109 C Fall 2014 Armen Zakarian Office: Chemistry Bldn 2217

Chem 109 C Fall 2014 Armen Zakarian Office: Chemistry Bldn 2217 Chem 109 C Fall 2014 Armen Zakarian ffice: Chemistry Bldn 2217! http://web.chem.ucsb.edu/~zakariangroup/courses.html! 1 Amino acids: Resolution of Racemates 2 Peptides/Proteins: Peptide Bonds - - - - peptides:

More information

Rapid and Reproducible Amino Acid Analysis of Physiological Fluids for Clinical Research Using LC/MS/MS with the atraq Kit

Rapid and Reproducible Amino Acid Analysis of Physiological Fluids for Clinical Research Using LC/MS/MS with the atraq Kit Rapid and Reproducible Amino Acid Analysis of Physiological Fluids for Clinical Research Using LC/MS/MS with the atraq Kit Fast, simple and cost effective analysis Many areas of biochemical research and

More information

Peptide Design Strategy: Basics, Optimization, and Application. Presented by: Tiffany Gupton Campolongo, Ph.D.

Peptide Design Strategy: Basics, Optimization, and Application. Presented by: Tiffany Gupton Campolongo, Ph.D. Peptide Design Strategy: Basics, Optimization, and Application Presented by: Tiffany Gupton Campolongo, Ph.D. Presentation overview 1 2 3 4 Introduction Peptide Design Basics Advanced Design Strategy Strategy

More information

1055 BIOTECHNOLOGY- priate, in specific monographs. DERIVED ARTICLES PEPTIDE MAPPING

1055 BIOTECHNOLOGY- priate, in specific monographs. DERIVED ARTICLES PEPTIDE MAPPING Official July 1, 2009 1055 Biotechnology-Derived Articles 1 1055 BIOTECHNOLOGY- priate, in specific monographs. DERIVED ARTICLES PEPTIDE MAPPING INTRODUCTION Peptide mapping is an identity test for proteins,

More information

The Behavior of Proteins:

The Behavior of Proteins: Mary K. Campbell Shawn O. Farrell http://academic.cengage.com/chemistry/campbell Chapter 7 The Behavior of Proteins: Enzymes, Mechanisms, and Control Paul D. Adams University of Arkansas The catalytic

More information

Polyacrylamide gel formation

Polyacrylamide gel formation Part II- Protein Identification PolyAcrylamide Gel Electrophoresis (PAGE) is the best method in protein identification, MW determination, DNA sequencing, protein-protein or protein-dna interaction etc

More information

Reading Assignment: pp , ,

Reading Assignment: pp , , Chapter 6 "Mechanisms of Enzymes" Reading Assignment: pp. 158-167, 171-176, 182-187. Problem Assignment: 1, 3, and 4. I. Introduction The first objective of this chapter is to obtain a conceptual understanding

More information

1. 1. Amino acids and proteins. 1: Biochemistry of macromolecules and metabolic pathways. Key terms

1. 1. Amino acids and proteins. 1: Biochemistry of macromolecules and metabolic pathways. Key terms 1. 1 Amino acids and proteins Key terms Polymer: A large molecule made from repeating units called monomers. Monomer: A molecule that is a basic unit; many monomers join together to make a polymer. Amino

More information

WORKING WITH PEPTIDES

WORKING WITH PEPTIDES WORKING WITH PEPTIDES 1 Synthetic custom peptides offer an increasingly affordable approach for exploring protein-protein interactions and more complex phenomena such as immune responses directed against

More information

Chapter 3 Amino Acids, Peptides, Proteins

Chapter 3 Amino Acids, Peptides, Proteins Chapter 3 Amino Acids, Peptides, Proteins Also start memorizing AA: names, structures, abbreviations, 1 letter codes, pka s of side chains 3.1 Amino acids Amino acid is monomer condensed into peptide (small)

More information

Peptide Synthesis. Technical Document. www.altabioscience.com. Why use AltaBioscience?

Peptide Synthesis. Technical Document. www.altabioscience.com. Why use AltaBioscience? Peptide Synthesis Technical Document AltaBioscience offers a custom peptide synthesis service certified to ISO 9001:2008. With a strong focus on scientific excellence and 40 years of expertise we work

More information

THE CHEMICAL SYNTHESIS OF PEPTIDES

THE CHEMICAL SYNTHESIS OF PEPTIDES TE EMIAL SYTESIS F PEPTIDES Peptides are the long molecular chains that make up proteins. Synthetic peptides are used either as drugs (as they are biologically active) or in the diagnosis of disease. Peptides

More information

Acidic amino acids: Those whose side chains can carry a negative charge at certain ph values. Typically aspartic acid, glutamic acid.

Acidic amino acids: Those whose side chains can carry a negative charge at certain ph values. Typically aspartic acid, glutamic acid. A Acidic amino acids: Those whose side chains can carry a negative charge at certain ph values. Typically aspartic acid, glutamic acid. Active site: Usually applied to catalytic site of an enzyme or where

More information

Protein Structure and Function

Protein Structure and Function Jones & Bartlett Learning, LL. T F SALE DISTIBUTI Protein Structure and Function SETI I APTE 2 APTE 3 Protein Structure Protein Function 27 Jones & Bartlett Learning, LL. T F SALE DISTIBUTI 2 Protein Structure

More information

AMINO ACIDS & PEPTIDE BONDS STRUCTURE, CLASSIFICATION & METABOLISM

AMINO ACIDS & PEPTIDE BONDS STRUCTURE, CLASSIFICATION & METABOLISM AMINO ACIDS & PEPTIDE BONDS STRUCTURE, CLASSIFICATION & METABOLISM OBJECTIVES At the end of this session the student should be able to, recognize the structures of the protein amino acid and state their

More information

Lecture 15: Enzymes & Kinetics Mechanisms

Lecture 15: Enzymes & Kinetics Mechanisms ROLE OF THE TRANSITION STATE Lecture 15: Enzymes & Kinetics Mechanisms Consider the reaction: H-O-H + Cl - H-O δ- H Cl δ- HO - + H-Cl Reactants Transition state Products Margaret A. Daugherty Fall 2004

More information

High-Throughput 3-D Chromatography Through Ion Exchange SPE

High-Throughput 3-D Chromatography Through Ion Exchange SPE High-Throughput 3-D Chromatography Through Ion Exchange SPE Application Note 205 Luke Roenneburg and Alan Hamstra (Gilson, Inc.) Introduction 2-dimensional (2-D) separation is the separation of a sample

More information

Chapter 7: The Behavior of Proteins: Enzymes, Mechanisms and Control

Chapter 7: The Behavior of Proteins: Enzymes, Mechanisms and Control Chapter 7: The Behavior of Proteins: Enzymes, Mechanisms and Control The behavior of allosteric enzymes The concerted and sequential model Control of enzyme activity by phosphorylation Zymogen The nature

More information

Application Note # MT-90 MALDI-TDS: A Coherent MALDI Top-Down-Sequencing Approach Applied to the ABRF-Protein Research Group Study 2008

Application Note # MT-90 MALDI-TDS: A Coherent MALDI Top-Down-Sequencing Approach Applied to the ABRF-Protein Research Group Study 2008 Bruker Daltonics Application Note # MT-90 MALDI-TDS: A Coherent MALDI Top-Down-Sequencing Approach Applied to the ABRF-Protein Research Group Study 2008 In the ABRF-PRG study 2008 [*] the ability to characterize

More information

Quality. Now Certified to ISO 9001:2008

Quality. Now Certified to ISO 9001:2008 Quality Now Certified to ISO 90012008 Quality Policy It is Peptides International's goal is to achieve complete customer satisfaction by addressing customer needs and delivering what we promise. The company

More information

ENZYMES FOR BIOCATALYSIS

ENZYMES FOR BIOCATALYSIS ENZYMES FOR BIOCATALYSIS for smarter chemical synthesis Graphical representation of alcalase molecule Rethink Tomorrow Proteases Biocatalysis Biocatalysis involves the implementation of natural catalysts,

More information

Application Note 177 INTRODUCTION

Application Note 177 INTRODUCTION Application Note 177 Separation of an Intact Monoclonal Antibody and Fractionation of Monoclonal Antibody Papain Digest Fragments Using Immobilized Metal Affinity Chromatography (IMAC) INTRODUCTION Monoclonal

More information

Master course KEMM03 Principles of Mass Spectrometric Protein Characterization. Exam

Master course KEMM03 Principles of Mass Spectrometric Protein Characterization. Exam Exam Master course KEMM03 Principles of Mass Spectrometric Protein Characterization 2010-10-29 kl 08.15-13.00 Use a new paper for answering each question! Write your name on each paper! Aids: Mini calculator,

More information

Prior to release, the following tests are carried out on each batch of the final substances.

Prior to release, the following tests are carried out on each batch of the final substances. Insulin Lispro C 257 H 383 N 65 O 77 S 6 Mol. Wt. 5808 28 B -L-Lysine-29 B -L-proline insulin (human) Insulin Lispro is a 2-chain peptide containing 51 amino acids. The A- chain is composed of 21 amino

More information

BOC334 (Proteomics) Practical 1. Calculating the charge of proteins

BOC334 (Proteomics) Practical 1. Calculating the charge of proteins BC334 (Proteomics) Practical 1 Calculating the charge of proteins Aliphatic amino acids (VAGLIP) N H 2 H Glycine, Gly, G no charge Hydrophobicity = 0.67 MW 57Da pk a CH = 2.35 pk a NH 2 = 9.6 pi=5.97 CH

More information

The Behavior of Proteins: Enzymes, Mechanisms, and Control. Chapter Seven

The Behavior of Proteins: Enzymes, Mechanisms, and Control. Chapter Seven The Behavior of Proteins: Enzymes, Mechanisms, and Control Chapter Seven Allosteric Enzymes Allosteric proteins: have quaternary structure arrangement which results from noncovalent interaction among subunits.

More information

Application Note. Determination of 17 AQC derivatized Amino acids in baby food samples. Summary. Introduction. Category Bio science, food Matrix

Application Note. Determination of 17 AQC derivatized Amino acids in baby food samples. Summary. Introduction. Category Bio science, food Matrix Application Note Determination of 17 AQC derivatized Amino acids in baby food samples Category Bio science, food Matrix Baby food Method UHPLC Keywords Proteinogenic amino acids, canonical amino acids,

More information

An algorithm for isoelectric point estimation

An algorithm for isoelectric point estimation An algorithm for isoelectric point estimation David L. Tabb Created 7/10/01 Updated 6/28/03 1 Introduction One of the most common techniques for separating mixtures of proteins is the two-dimensional polyacrylamide

More information

Molecular Cell Biology. Prof. D. Karunagaran. Department of Biotechnology. Indian Institute of Technology Madras

Molecular Cell Biology. Prof. D. Karunagaran. Department of Biotechnology. Indian Institute of Technology Madras Molecular Cell Biology Prof. D. Karunagaran Department of Biotechnology Indian Institute of Technology Madras Module 5 Methods in Cell Biology (Methods to Manipulate Protein, DNA and RNA and Methods to

More information

Methods in Enzymology. Volume XL VII. Enzyme Structure. Part E. C. H. W. Hirs. Serge N. Timasheff

Methods in Enzymology. Volume XL VII. Enzyme Structure. Part E. C. H. W. Hirs. Serge N. Timasheff Methods in Enzymology Volume XL VII Enzyme Structure Part E C. H. W. Hirs Serge N. Timasheff CONTRIBUTORS TO VOLUME XLVII PREFACE VOLUMES IN SERIES ix xii i XV Section I. Amino Acid Analysis 1. Subnanomole-Range

More information

Polypeptides and Proteins

Polypeptides and Proteins Polypeptides and Proteins These molecules are composed, at least in part, of chains of amino acids. Each amino acid is joined to the next one through an amide or peptide bond from the carbonyl carbon of

More information

PROTEINS STRUCTURE AND FUNCTION (DR. TRAISH)

PROTEINS STRUCTURE AND FUNCTION (DR. TRAISH) Introduction to Proteins - Proteins are abundant and functionally diverse molecules - They participate in cell regulation at all levels - They share a common structural feature: all are linear polymers

More information

Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a

Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a Optimal Conditions for F(ab ) 2 Antibody Fragment Production from Mouse IgG2a Ryan S. Stowers, 1 Jacqueline A. Callihan, 2 James D. Bryers 2 1 Department of Bioengineering, Clemson University, Clemson,

More information

AMINO ACID SEQUENCE HOMOLOGY BETWEEN HUMAN AND BOVINE LOW MOLECULAR WEIGHT FOLATE BINDING PROTEIN ISOLATED FROM MILK

AMINO ACID SEQUENCE HOMOLOGY BETWEEN HUMAN AND BOVINE LOW MOLECULAR WEIGHT FOLATE BINDING PROTEIN ISOLATED FROM MILK Carlsberg Res. Commun. Wol. 47, p. 371-376, 1982 AMINO ACID SEQUENCE HOMOLOGY BETWEEN HUMAN AND BOVINE LOW MOLECULAR WEIGHT FOLATE BINDING PROTEIN ISOLATED FROM MILK by IB SVENDSEN Carlsberg Laboratory,

More information

1. The diagram below represents a biological process

1. The diagram below represents a biological process 1. The diagram below represents a biological process 5. The chart below indicates the elements contained in four different molecules and the number of atoms of each element in those molecules. Which set

More information

TECHNICAL BULLETIN. ProteoMass Peptide & Protein MALDI-MS Calibration Kit. Catalog Number MSCAL1 Store at Room Temperature

TECHNICAL BULLETIN. ProteoMass Peptide & Protein MALDI-MS Calibration Kit. Catalog Number MSCAL1 Store at Room Temperature ProteoMass Peptide & Protein MALDI-MS Calibration Kit Catalog Number MSCAL1 Store at Room Temperature TECHNICAL BULLETIN Description This kit provides a range of standard peptides and proteins for the

More information

Shu-Ping Lin, Ph.D. E-mail: splin@dragon.nchu.edu.tw

Shu-Ping Lin, Ph.D. E-mail: splin@dragon.nchu.edu.tw Amino Acids & Proteins Shu-Ping Lin, Ph.D. Institute te of Biomedical Engineering ing E-mail: splin@dragon.nchu.edu.tw Website: http://web.nchu.edu.tw/pweb/users/splin/ edu tw/pweb/users/splin/ Date: 10.13.2010

More information

Introduction to Proteins; Amino Acids, the Building Blocks of Proteins

Introduction to Proteins; Amino Acids, the Building Blocks of Proteins Introduction to Proteins; Amino Acids, the Building Blocks of Proteins Reading: Berg, Tymoczko & Stryer: Chapter 2, pp. 25-34 Appendix to Chapter 2, pp. 60-61 (visualizing protein structures) Review General

More information

Laboration 1. Identifiering av proteiner med Mass Spektrometri. Klinisk Kemisk Diagnostik

Laboration 1. Identifiering av proteiner med Mass Spektrometri. Klinisk Kemisk Diagnostik Laboration 1 Identifiering av proteiner med Mass Spektrometri Klinisk Kemisk Diagnostik Sven Kjellström 2014 kjellstrom.sven@gmail.com 0702-935060 Laboration 1 Klinisk Kemisk Diagnostik Identifiering av

More information

INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE Q6B. Current Step 4 version

INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE Q6B. Current Step 4 version INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE ICH HARMONISED TRIPARTITE GUIDELINE SPECIFICATIONS: TEST PROCEDURES AND ACCEPTANCE

More information

Size Exclusion Chromatography

Size Exclusion Chromatography Size Exclusion Chromatography TOYOPEARL Resins for SEC TOYOPEARL TOYOPEARL TOYOPEARL HW-55 TOYOPEARL HW-65 TOYOPEARL TOSOH BIOSCIENCE TOSOH BIOSCIENCE LLC 56 Keystone Drive Montgomeryville, PA 896-967

More information

Standard practices for Fmoc-based solid-phase. peptide synthesis in the Nowick laboratory. (Version 1.6.1)

Standard practices for Fmoc-based solid-phase. peptide synthesis in the Nowick laboratory. (Version 1.6.1) Standard practices for Fmoc-based solid-phase peptide synthesis in the Nowick laboratory (Version 1.6.1) Adam G. Kreutzer and Patrick J. Salveson E-mail: Contents Contributions to this guide 3 General

More information

Solution problem 13: Absorption of Light by Molecules

Solution problem 13: Absorption of Light by Molecules Solution problem 13: Absorption of Light by Molecules 13.1 A = εcd = 1.5 10 5 mol -1 L cm -1 4 10-6 mol L -1 10-4 cm = 6 10-5 Since A = log(p 0 /P), the ratio P/P 0 is 0.999862. This is the percentage

More information

Nafith Abu Tarboush DDS, MSc, PhD natarboush@ju.edu.jo www.facebook.com/natarboush

Nafith Abu Tarboush DDS, MSc, PhD natarboush@ju.edu.jo www.facebook.com/natarboush Nafith Abu Tarboush DDS, MSc, PhD natarboush@ju.edu.jo www.facebook.com/natarboush α-keratins, bundles of α- helices Contain polypeptide chains organized approximately parallel along a single axis: Consist

More information

Enzymes Enzyme Mechanism

Enzymes Enzyme Mechanism Mechanisms of Enzymes BCMB 3100 Chapters 6, 7, 8 Enzymes Enzyme Mechanism 1 Energy diagrams Binding modes of enzyme catalysis Chemical modes of enzyme catalysis Acid-Base catalysis Covalent catalysis Binding

More information