Protein Analysis Lecture Notes PDF

CH4306 Bioanalytical Techniques Protein Analysis COLLEGE OF ENGINEERING SCHOOL OF CHEMISTRY, CHEMICAL ENGINEERING AND BIOTECHNOLOGY Kit Wayne Chew [email protected] Course schedule and content Week Date Topics 8 10 October 2023 Protein Analysis (Chapter 1 + 8) 9 17 October 2023 Ch...

CH4306 Bioanalytical Techniques Protein Analysis COLLEGE OF ENGINEERING SCHOOL OF CHEMISTRY, CHEMICAL ENGINEERING AND BIOTECHNOLOGY Kit Wayne Chew [email protected] Course schedule and content Week Date Topics 8 10 October 2023 Protein Analysis (Chapter 1 + 8) 9 17 October 2023 Chromatography-1 (Chapter 2) 10 24 October 2023 Chromatography-2 (Chapter 2) 11 31 October 2023 Mass Spectrometry-1 (Chapter 4) 12 7 November 2023 Mass Spectrometry-1 (Chapter 4) 13 14 November 2023 Special Topics in Enzymology Andreas Manz, Petra Dittrich, Nicole Pamme, and Dimitri Iossifidis. Bioanalytical Chemistry, Imperial College Press (2015, 2nd Edition). Lecture notes from Dr. Chen Ming-Hsu, Dr. Guan Xueli, and Dr. Dave Wei. Week 9. Protein analysis A B C • Chemistry of amino acids, peptides and proteins ➢Amino acids structure, classification and properties ➢Protein structure, classification and properties • Protein purification ➢Protein extraction ➢Protein separation ➢Concentration measurement • Protein sequencing ➢Subunit determination ➢Amino acid composition determination ➢Peptide cleavage ➢Amino acid sequencing and peptide ordering ➢Disulfide bond cleavage and assignment Part A Protein discovery and definition ➢ In 1839, Dutch chemist GJ Mulder while investigating substances in milk and eggs observed that they seemed to have same empirical formula (C20H31N5O12). ➢ Swedish chemist JJ Berzelius suggested that these substances should be called proteins. ➢ Berzelius thought them to be most important of biological substances. (Proteios means “primary” or “pre-eminent”). ➢ Modern definition: Proteins are nitrogenous “macromolecules” composed of many amino acids. Dr. Chen Ming-Hsu Gerardus Johannes Mulder Chemist (1802 – 1880) Jöns Jacob Berzelius Chemist (1779–1848) Biological function of protein ➢ Main structural components of the cytoskeleton. ➢ Biochemical catalysts known as enzymes. ➢ Immunoglobulins serve as the first line of defense against bacterial and viral infection. ➢ Some hormones are proteins. ➢ Structural proteins, actin and myosin, help in the movement of muscle fiber. ➢ Proteins present in cell membrane, cytoplasm, and nucleus of the cell act as receptors. ➢ Transport proteins carry specific substances across membrane. ➢ Storage proteins bind and store certain substances, e.q. iron. ➢ Proteins can be catabolized to supply energy. Biochemistry (Chatterjea and Shinde, 2012) https://en.wikipedia.org Amino acid Amino acids are organic compounds that contain amino (−NH2) and carboxylic acid (−COOH) functional groups, along with a side chain (R group) specific to each amino acid. 3 4 R H C H2N α COOH L-isomer or S-form https://en.wikipedia.org/wiki/Amino_acid HOOC C 2 1 Fig. Amino acid codon wheel. R D-isomer or R-form H NH2 20 Common amino acids Nonpolar, aliphatic R groups Gly, G Ala, A Pro, P Aromatic R groups Val, V Ser, S Phe, F Tyr, Y Trp, W Tyr and Trp are relatively hydrophilic than Phe; A280= ε𝑐𝑙 Leu, L Ile, I Negatively charged Polar, uncharged R groups R groups Asp, D Cys, C Glu, E Positively charged R groups Asn, N Met, M Hydrophobic: side chain R contains C, H only Gln, Q Hydrophilic: side chain R contains C, H, N and O Lys, K https://en.wikipedia.org Thr, T Arg, R His, H Non-standard amino acids Non-standard amino acids Structure Function β-alanine A constituent in coenzyme A and carnosine. Taurine Found in bile acids. Ornithine Intermediates in the urea cycle. ϒ-aminobutyric acid (GABA) A neurotransmitter produced from the glutamic acid Dr. Chen, Ming-Hsu Zwitterion form of amino acid very high m p, . strong for A zwitterion, also called an inner salt or dipolar ion, is a molecule having separate positively and negatively charged functional groups. R H C H2N COOH -H+ -H+ H R H R C C + H3N COO j H2N COO- H R C + H3N D COOH Low extra 0 2 https://en.wikipedia.org/wiki/Amino_acid 4 PH M 6 8 10 12 Isoelectronic point (pI) of amino acids acid α-carboxyl α-amino -COOH -N+H3 Conjugate base -COO-NH2 2.34 pK1 9.60 pK2 𝑝𝐾1 + 𝑝𝐾2 𝑝𝐼 = 2 Amino acids with ionizable R Ionizable Group Acid α-carboxyl -COOH ↔ −COO− 3.1 α-amino -N+H3 ↔ −NH2 8.0 Aspartic acid -COOH ↔ −COO− 3.9 Glutamic acid -COOH ↔ −COO− 4.1 Histidine Cysteine Lysine Tyrosine Arginine Conjugate Base H + ↔ pKa 6.0 -SH ↔ -S- 10.54 -N+H3 ↔ −NH2 10.8 O- OH↔ NH N+H2 ↔ 10.9 12.5 ↓ 3 pla= platykantyka, pI of amino acids with ionizable R acid α-carboxyl -COOH 𝑝𝐼 = Conjugate base 1.99 -COO- pK1 α-amino -N+H3 -NH2 9.9 pK2 R-carboxyl -COOH -COO- 3.9 pK3 acid α-carboxyl -COOH Conjugate base 2.16 -COO- pK1 α-amino -N+H3 -NH2 9.06 pK2 R-amino -N+H3 -NH2 10.54 pK3 = 𝑝𝐾1 + 𝑝𝐾3 2 1.99 + 3.9 2 = 2.9 𝑝𝐾2 + 𝑝𝐾3 𝑝𝐼 = 2 9.06 + 10.54 = 2 =9.8 Protein pI calculation 1. Estimate the pH at which the net charge on the protein would be zero. 2. Find the average of the two pKa values directly above and directly below the estimate. The isoelectronic point will be halfway between these two pKa. Find the pI of Asp-Gly-Glu. pKa=4.1 COO- pKa=3.9 COOCH2 COO- H Assume pI=7.0, H CH2 CH2 H3N-CH-CO-NH- CH -CO-NH-CH -COO- CH2 CH2 H2N-CH-CO-NH- CH -CO-NH-CH-COOpKa=3.1 pKa=8.0 CH2 COO- COOH COOH Assume pI=3.5, CH2 H CH2 CH2 H3N-CH-CO-NH- CH -CO-NH-CH -COOpI= 3.9+3.1 2 = 3.5 Isoelectronic point of proteins Isoelectric focussing technique In-class Discussion-1 ➢ Which of the common amino acids is achiral ? ➢ pK1 of arginine is 1.82, pK2 is 8.99 and pK3 is 12.48. What is the expected pI of arginine? ➢ Imagine you are working in a microbiology lab. To prepare a stock solution of 20 amino acids (equal molar), what information do you need? Peptides - actual protein is a . combination of hondred differentamino acid chains Peptides are linear sequence of amino acids linked together by peptide/amide bonds. + H2O Peptide bond formation is not thermodynamic favourable require activation in order to create the bond then become , thermodynamically partorable ↓ Enzyme can activation 3 Biochemistry (Hames and Hooper, 2005) Biochemistry (Nelson and Cox, 2012) Resonance structure of the peptide bond. Peptide units within a polypeptide. Biological Functions of Peptides Artificial sweetener Hormones Oxytocin cysteine-tyrosine-isoleucine-glutamine-asparaginecysteine-proline-leucine-glycine-amide Insulin Biological Functions of Peptides Antimicrobial agent The human beta-defensin-1 (hBD-1) is a short basic peptide of 36 amino acid residues. It contains six cysteines forming three intramolecular disulfide bonds. The molecular mass of hBD-1 is 3928.6 Da. Mechanisms of action of defensin. The defensin with its positive charge is attached by electrostatic attraction to the membrane of the pathogen forming pores. https://www.intechopen.com/chapters/63967 In-class Discussion-2 Here are two tetrapeptides, please indicate the sequence of amino acid residues: Ala-Tyr-Asp-Gly Ser-Ala-Cys-Gly Proteins Amino acids Peptide Protein Primary structure: the sequence ➢ Primary structure is the linear sequence of amino acids held together by peptide bonds. ➢ The free –NH2 group of the terminal amino acid is called N-terminal end, the free –COOH group is called C-terminal end. ➢ It is traditional to number the amino acids from N-terminal end. ➢ Even one amino acid change can affect the protein’s overall structure and function. Biochemistry (Nelson and Cox, 2012) https://www.open.edu Protein Secondary Structure ➢ The peptide chain can form a three dimensional secondary structure by folding or coiling. ➢ It results from the steric relationship between amino acids located relatively near each other. ➢ The main linkages involve in the secondary structure formation are hydrogen bonds. Hydrogen bonds: These are weak, low energy non-covalent bonds sharing a single hydrogen by two electron negative atoms such as O and N. Biochemistry (Chatterjea and Shinde, 2012) https://courses.lumenlearning.com; https://www.quora.com Protein secondary structure: α-helix ➢ A peptide chain forms regular helical coils called alpha helix. ➢ The structure is stabilized by hydrogen bonds between carbonyl O of nth amino and amide H of n+4th amino acid residues. ➢ Only right handed α-helices are found in protein. ➢ One complete run: 3.6 amino acids ➢ Small and uncharged amino acid residues (alanine, leucine, phenylalanine) are often found in α-helix. Biochemistry (Chatterjea and Shinde, 2012) Biochemistry (Hames and Hooper, 2005) Protein secondary structure: β-pleated sheet ➢ Beta-pleated sheet structure is formed when hydrogen bonds are formed between the carbonyl oxygens and amide hydrogens of two or more adjacent peptides. ➢ Interchain bonding. ➢ Parallel or antiparallel. ➢ Glycine, serine, and alanine are common. ➢ Beta-keratin in silk fibroin and spider web. Biochemistry (Chatterjea and Shinde, 2012) Biochemistry (Hames and Hooper, 2005) Protein secondary structure: β-turn ➢ Polypeptide chain reverse direction. ➢ Carbonyl oxygen of first residue is hydrogen bonded to the amide hydrogen of the fourth residue. ➢ It is 180° turn involving four amino acid residues. ➢ Gly and Pro residues often occur in β-turn structure. ➢ Often found at the surface of a protein. Biochemistry (Nelson and Cox, 2012) Proline isomers Protein tertiary structure ➢ Polypeptide chain with secondary structure may be further folded/twisted to form tertiary structure. ➢ Tertiary structure is constituted by steric relationships between the amino acids located far apart but brought together by folding. ➢ The conformation is called as native protein. Bonds Hydrophobic interactions Between non-polar side chains of amino acids Hydrogen bonds Formed by the polar side chains Ionic interactions Formed between charged side chains Van der Waal forces Instantaneous dipole induced forces between non-polar side chains The S-S bonds between distant cysteine residues Disulfide bonds Biochemistry (Murray, Bender, Botham, Kennelly, Rodwell and Weil, 2009) Biochemistry (Hames and Hooper, 2005); Biochemistry (Chatterjea and Shinde, 2012) Cysteine Cystine https://cbm.msoe.edu/teachingResources/proteinStructure/as sets/video/hydrophobic.mp4 https://cbm.msoe.edu/teachingResources/proteinStructure/as sets/video/hydrophillic.mp4 https://cbm.msoe.edu/teachingResources/proteinStructure/as sets/video/positiveNegative.mp4 https://cbm.msoe.edu/teachingResources/proteinStructure/as sets/video/cysteine.mp4 Protein quaternary structure ➢ When a protein consists of two or more peptide chains, it is referred to as the quaternary structure. ➢ Both non-covalent or covalent cross-links. ➢ Each consistent peptide is called as monomer or subunit. homo-trimer tetramer Hemoglobin Protein denaturation ➢Denaturation is defined as the disruption of the secondary, tertiary, and quaternary organization due to cleavage of non-covalent bonds. ➢ Primary structure is not affected by denaturation. ➢ Loss of protein structure results in loss of function. Agents that cause denaturation Types Physical agents Heat, UV light, ultrasound, and high pressure Chemical agents Organic solvents, acids/alkalis, urea, and detergents Biochemistry (Nelson and Cox, 2012); Biochemistry (Chatterjea and Shinde, 2012) https://ib.bioninja.com.au In-class Discussion-3 1 Please label 1-4 R group interactions that contribute to tertiary protein structure. 1 2 2 4 3 4 3 Analytical techniques corresponding to different structural levels of protein Structural Element Information Gained Analytical Technique Primary The specific sequence of amino acids in polypeptides Edman degradation Mass Spectrometry (MS) Secondary Coiling and folding of polypeptide chain e.g. α-helix, β-sheets, and random coils Circular Dichroism (CD) Infrared Spectroscopy (IR) Tertiary Overall 3D Shape or form of a single polypeptide X-Ray Diffraction Crystallography (XRD) Nuclear Magnetic Resonance (NMR) Quaternary Overall 3D structure of proteins composed of Size Exclusion Chromatography (SEC) two or more polypeptide chains Dynamic Light Scattering (DSC) Analytical Ultra Centrifugation (AUC) 3 2 Part B Protein purification The aim of protein purification is to isolate one particular protein from all the others in the starting material. A combination of fractionation techniques is used that exploits the solubility, size, charge, hydrophobicity or specific binding affinity of the protein of interest. Material selection Homogenization & solubilization Stabilization of proteins Assays of proteins Fractionation Fig. Typical sequence for the purification of a protein. Chromatographic procedures Purity determination 3 3 Protein purification ➢ Material selection ➢ Crude intracellular protein extraction ➢ Protein fractionation and separation ➢ Measuring protein concentration ➢ Electrophoresis and gel staining Choice of raw material ➢ Selection of the protein source (make the right choice!) https://marketfresh.com.sg ➢ A source that is relatively rich in the protein of interest and which is readily available. 100 Fig. Yield from multi-step purifications. Fig. Location and approximate numbers of proteins in E.coli. Protein purification (Jason, 2011) Protein purification handbook (Amersham Biosciences) 3 5 Preparation of crude extract ➢ Liquid extracellular proteins can be directly applied into separation. ➢ Optimization of extraction conditions from a solid source. ➢ Breaking down cells—homogenization. ➢ A compromise between recovery and purity. ➢ Things to consider: Hand-operated or motor-driven Waring blender 1. Equipment type 2. Extraction medium Ultrasound 3. Temperature (2-8°C) 4. Time Vibrating bead mill Manton-Gaulin homogenizer Fig. Equipment used for breaking up cells to obtain an extract. Protein purification (Scopes, 1994) Protein purification (Jason, 2011) Mild Techniques for protein extraction ➢ Chemical Repeated freezing and thawing ➢ Mechanical Moderate ➢ Physical Vigorous 3 7 Extraction medium ➢ To stabilize the protein in the crude extract. ➢ To release the protein from the cells or tissue. ➢ The following factors have to be taken into consideration: Table Buffer salts used in protein work. 1. pH 2. Buffer salts 3. Detergents 4. Reducing agents 5. Chelators or metal ions 6. Proteolytic inhibitors 7. Bacteriostatics Protein purification (Scopes, 1994) Protein purification (Jason, 2011) Extraction medium-detergents ➢ The desired protein is bound to particles or aggregated due to the hydrophobic interactions. ➢ Using detergents can break the hydrophobic interactions. ➢ Just needed at the extraction stage. ➢ Detergent concentrations below critical micelle concentration (CMC should be used). Protein purification (Jason, 2011) https://www.labome.com Table Detergents used for solubilization of proteins. Extraction medium-reducing agents ➢ Protein oxidation could be a concern during purification process. ➢ The thiol group (R-SH) is susceptible to oxidation. ➢ Protein thiol group can be protected by reducing agents such as DTE, DTT, and mercaptoethanol. ➢ Ascorbic acid is sometimes added to plant extract to avoid oxidation or miscoloration. Protein purification (Jason, 2011) Table Reducing agents. Extraction medium-chelators and metal ions ➢ Presence of metal ions in crude extract could be both beneficial and harmful. ➢ Ions such as Na+, K+, Ca2+, Mg2+ and Fe3+ may increase the stability of protein. ➢ Many enzymes require specific metal ions for activity. ➢ Heavy metal ions such as Ag+, Cu2+, Pb2+ and Hg2+ can enhance the oxidation reaction. ➢ Chelating agent such as ethylenediamine tetraacetic acid (EDTA) can be used to remove heavy metals. Fig. Ethylene glycol tetraacetic acid (EGTA). https://pharmafactz.com https://www.goldbio.com Extraction medium-proteolytic inhibitors ➢ Proteases can degrade targeted protein during purification process. ➢ Maintain at low temperatures. ➢ Add protease inhibitors to the crude extract. ➢ Sometimes need a mixture of protease inhibitors. Table Proteolytic inhibitors. Table Classification of proteases. Protein purification (Jason, 2011) Baek and Choi (2008) Extraction medium-bacteriostatics ➢ Take precautions to avoid bacterial growth in protein solutions. ➢ Prepare sterile filtered buffer solutions. ➢ Phosphate, acetate, and carbonate buffers at neutral pH support bacterial growth. stable& the sect ↓ need to see ipurtein is ➢ pH value below 3 or above 9 can prevent bacterial growth. ➢ Antimicrobial agents can be added. Antimicrobial agents Concentration Sodium azide 0.001M Merthiolate 0.005% Alcohols-butanol 1% Protein purification (Jason, 2011) Krivoruchko et al. (2014) Protein separation: precipitation protein is slightly sooble in water : - need to gothro protein precipitation step ➢ Precipitation is used as preliminary steps for initial fractionation. ➢ Separation method based on solubility. ➢ Distribution of hydrophilic and hydrophobic residues at the surface of protein determines the solubility. ➢ Precipitation can be achieved by adding salts, organic solvents, or polymers, or by adjusting the pH and temperature. ~ assistin precipitation ➢ Salts used as precipitation agents are: Anions: PO43-, SO42-, CH3COO-, Cl-, Br-, NO3-, ClO3-, I-, SCN- Cations: NH4+, K+, Na+, Guanidine C(NH2)3+ added [retsaturated Make sore that an appropriate and is . solution as they can change the phopthe Protein purification (Scopes, 1994) Protein purification (Jason, 2011) Table Precipitation agents. Protein precipitation: ammonium sulphate ➢ Salting out at high salt concentrations. ➢ Largely depend on the hydrophobicity of the protein. ➢ Dissolved salt ions compete with protein for water molecules. ➢ Causing protein to fold, aggregate and eventually precipitate. Protein purification (Scopes, 1994) Campbell, 1996. Table Trial fractionations with ammonium sulphate. Protein precipitation: organic solvents ➢ Addition of a miscible solvent (ethanol or acetone) can induce protein precipitation. ➢ Reduction in water activity & bulk displacement of water. ➢ The larger the protein molecule, the lower percentage of solvent required to precipitate it. ➢ The organic solvent may break protein intramolecular interactions and cause denaturation. Protein purification (Scopes, 1994) Protein purification: Dialysis ➢ Proteins can be separated from small molecules by dialysis through a semi-permeable membrane. ➢ Often used to remove salts and ammonium sulphate. ➢ At equilibrium, the concentration of small molecules inside a dialysis bag will be equal to outside. ➢ Several changes of the solution are often required. https://www.thermofisher.com Starting point Biochemistry (Hames and Hooper, 2005) At equilibrium https://www.fishersci.com https://uk.vwr.com 4 7 Protein separation by chromatographic steps ➢ Gel filtration chromatography (size exclusion chromatography) ➢ Ion exchange chromatography ➢ Affinity chromatography ➢ Design a logical sequence of chromatographic steps. ➢ Parameters to consider: 1. Sample volume 2. Protein concentration and viscosity of sample 3. The degree of purity of protein extract 4. Presence of nucleic acids, pyrogens, and proteolytic enzymes 5. Efficiency of each method in separating protein and contaminants. Protein purification (Jason, 2011) Measuring overall protein concentration ➢ It is important to quantify the total amount of protein present in the extracts and fractions. ➢ The percent recovery and the degree of purification can be calculated. ➢ Most widely used methods are: 1. UV absorption at 280 nm 2. Lowry-Folin-Ciocalteau reagent 3. Biuret-alkaline copper reagent 4. Dye binding 5. Bis-cinchonic acid (BCA) reagent Protein purification (Scopes, 1994) Table Advantages and disadvantages of methods of protein determination. Protein quantification: UV absorption ➢ Aromatic amino acids, tyrosine and tryptophan, generate ultraviolet absorbance at 280 nm. ➢ Simple method, no reaction is needed. ➢ Must not contain other components with absorbance at 280 nm. http://www.kemtrak.com/applications_pharmaceutical.html?panel=5 https://www.biotek.com 5 0 Protein quantification: Biuret method ➢ Use a strong alkaline copper reagent. ➢ Detecting the presence of peptide bonds. ➢ Produce a purple coloration with protein. ➢ Detected at 540 nm. large ( can https://www.analiticaweb.com.br https://en.wikipedia.org , ofpeptide bond detect protein outin system need calibration core iRnot is useless . 5 1 Protein quantification: Lowry method ➢ Lowry assay is widely used and often-cited (1951). ➢ A combination of the biuret method and the reaction of the Folin-Ciocalteau reagent. ➢ Folin-Ciocalteau reagent reacts with aromatic group of tyrosine and tryptophan residues . ➢ Form a strong, dark, blue color with proteins. ➢ Detected at 500 - 750, usually 660 nm. Folin-Ciocalteau reagent ➢ Many modifications have been reported. Sodium tungstate (Na2WO4) Sodium molybdate (Na2MoO4) Phosphoric acid (H3PO4) Hydrochloric acid (HCl) Lithium sulphate (Li2SO4) Bromine (Br) Protein purification (Scopes, 1994) https://en.wikipedia.org Protein quantification: bicinchonic acid (BCA) method ➢ Patented by Pierce Chemicals (Rockford, IL). ➢ Mixed reagent containing both copper and BCA reagent. ➢ Compatible with samples that contain 5% detergents. ➢ BCA reagent is 100 times more sensitive than biuret reagent. ➢ Detected at 562 nm. https://en.wikipedia.org/wiki/Bicinchoninic_acid_assay https://www.thermofisher.com/; https://en.wikipedia.org/ https://www.labome.com/method/Protein-Quantitation.html Protein quantification: dye binding ➢ The Bradford protein assay. ➢ Developed by Marion Bradford in 1976. ➢ Coomassie Brilliant Blue G-250 ➢ When the dye binds to protein, blue color complex is formed. ➢ Detected at 595 nm. https://www.labome.com/method/Protein-Quantitation.html Georgio et al. (2008) 5 4 Protein analysis for purity : electrophoresis Electrophoresis: ➢ When placed in an electric field, molecules with a net charge will move towards one electrode or the other. ➢ Each protein has a specific electrophoretic mobility, m, which determines its migration velocity, v, in an electric field E (measured in V/cm), and is therefore decisive for the separation. 𝑣=𝑚×𝐸 ➢ Electrophoretic mobility is dependent on the net charge and the size of the molecule. Fig. Separation principles of electrophoresis. Protein purification (Jason, 2011) 5 5 Types of electrophoresis systems Vertical and horizontal systems (A) System for gel rods in glass tubes. (B) Vertical slab gel system without cooling. (C) Vertical system for two or four slab gels. (D-a) Horizontal flatbed system-with paper wicks. (D-b) Horizontal flatbed system-with polyacrylamide strips. (D-c) Horizontal flatbed system-with filter paper strips. (E) Horizontal minigels-with agarose buffer strips. Protein purification (Jason, 2011) 5 6 Gel media Agarose gels: ➢ Used for separating protein over 800 kDa. ➢ Pore size from 150 nm (1% agarose) to 500 nm (0.16% agarose). Polyacrylamide gels: ➢ Mechanically and chemically more stable. ➢ Chemical copolymerization of acrylamide, crosslinker and initiator. ➢ Standard polymerization reaction needs: Buffer solution (Tris-HCl) Acrylamide/bisacrylamide solution N,N,N’N’-tetramethylethylene diamine (TEMED) 10% ammonium persulfate (APS) Protein purification (Jason, 2011) Fig. SEM figure of 2% agarose gel (Anders Medin, 1995). 5 7 Casting polyacrylamide gels ➢ Two layers of gels. - stacking Low S ↳werpft Restring Higher pl : . : ➢ Protein molecules migrate through the large pore stacking gel and then are separated in the resolving gel. ➢ Form a tight band, which improves resolution. Table Recipes for stacking and resolving gels. https://www.bio-rad.com 5 8 SDS-PAGE ➢ Laemmi (1970) incorporated sodium dodecyl sulphate (SDS) into protein electrophoresis system. ➢ Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) ➢ Proteins become fully denatured and dissociate from each other. ➢ Didulfide bridges can be opened using 2mercaptoethanol. ➢ SDS binds protein non-covalently and generate negative charges. The negative charge per mass unit of the peptide is constant. ➢ Bind at a ratio of 1.4 g SDS per 1 g of protein. ➢ The migration of SDS-bound protein solely depends on the molecular size. http://library.open.oregonstate.edu https://www.bio-rad.com 5 9 Gel staining methods 1. Coomassie brilliant blue 2. Reversible imidazole zinc staining ➢ Stains all areas of gel ➢ Can be easily removed 3. Silver staining ➢ High sensitivity (picogram range) Imidazole zinc staining 4. Fluorescent staining ➢ High sensitivity ➢ Deep Purple (from GE Healthcare) ➢ SYPRO Ruby (from Invitrogen) Fluorescent staining http://library.open.oregonstate.edu https://www.bio-rad.com Silver staining Part C Protein sequencing ➢ To determine the primary structure. ➢ The primary structure must be identified before elucidation of the secondary, tertiary and quaternary structure. ➢ Example: Sickle cell disease (a single amino acid substitution) ➢ Protein sequencing was pioneered by Frederick Sanger. ➢ Sanger determined the sequence of bovine insulin (1953). ➢ For this accomplishment, Sanger was awarded Nobel Prize of Chemistry in 1958. Biochemistry (Nelson and Cox, 2012) http://sickle.bwh.harvard.edu/scd_background.html Frederick Sanger (1918-2013) Nobel Prize in Chemistry (1958, 1980) Strategy for revealing the primary structure ➢ The number of distinct polypeptide chains (subunits) in the protein must be determined. ➢ Disulfide bond, must be cleaved. ➢ The amino acid composition of each polypeptide can be determined. ➢ Subunits must be fragmented into sets of smaller peptides by specific cleavage reactions. ➢ Each fragment is sequenced by employing Edman degradation. ➢ The sequence of each subunit can be put together by comparing overlaps of the different fragments. ➢ The whole structure, including disulfide bonds, can be determined. Andreas Manz, Petra Dittrich, Nicole Pamme, and Dimitri Iossifidis. Bioanalytical Chemistry. Protein sequencing Step 1. End group analysis: how many different types of subunits Step 2. Cleavage of the disulfide bonds Step 3. Separation, purification, and characterization of the subunits Step 4. Determination of amino acid composition Step 5. Specific peptide cleavage reactions Step 6. Sequence determination Step 7. Ordering the peptide fragments Step 8. Assignment of disulfide bond positions Step 1. End group analysis: how many different types of subunits ➢ Determine the number of different polypeptide chains (subunits) in the protein. ➢ Analyze the N-terminal and C-terminal residue (the end groups). ➢ By identifying the end groups, can establish the number of distinct polypeptides in a protein. ➢ Insulin has equal amounts of N-terminal residues Phe and Gly---indicate it has equal numbers of two distinct polypeptide chains. Bioanalytical Chemistry (Manz et al., 2015) Biochemistry (Nelson and Cox, 2012) N-terminus identification There are several methods to determine the Nterminal residue of a polypeptide: Dansyl chloride method ➢ 1-dimethyl-amino-naphthalene-5- sulfonyl chloride ➢ Reacts with primary amines, yield dansylated polypeptides. ➢ N-terminal residue is released in the form of dansylamino acid after acid hydrolysis. ➢ Exhibit an intense yellow florescent. N-terminus identification Phenyl isothiocyanate (PITC) and Edman degradation ➢PITC reacts with N-terminal amino group (alkaline condition). ➢ Form PTC polypeptide. ➢N-terminal residue is cleaved and form thiazolinone derivative. ➢ The remainder of the peptide keeps intact. ➢Thiazolinone-amino acid is converted to more stable phenylthiohydantoin (PTH). ➢ PTH can be identified by HPLC (UV 269 nm). ➢Originally described by Pehr Edman (1950) and become automated in 1967. Bioanalytical Chemistry (Manz et al., 2015) 56 Edman degradation mechanism https://www.youtube.com/watch?v=7nubm99YOyw N-terminus identification Aminopeptidase ➢ Enzymes that catalyze the cleavage of amino acids from the N-terminus. ➢ Exopeptidase ➢ Only a limited use for the determination of amino acid sequence. ➢ Some amino acids may be more resistant to the enzyme than the others. ➢ Different cleavage rates. http://www.worthington-biochem.com Gonzales and Robert-Baudouy (1996) 1. endopeptidase; 2. aminopeptidase; 3. carboxypeptidase C-terminus identification ➢ For C-terminus identification, there is no reliable method comparable to Edman degradation. ➢ Can be done using carboxypeptidase. ➢ Carboxypeptidases exhibit selectivity towards the amino acid side chains. ➢ C-terminal residues with a proceeding Pro residues are not subject to cleavage by carboxypeptidase A & B. ➢ The C-terminal residues are not cleaved at the same rate. ➢ Certain residues are resistant to carboxypeptidases. Table Specificities of various exopeptidase. C-terminus identification by hydrazinolysis ➢ Polypeptide is treated with anhydrous hydrazine at 90°C for 20-100 h. ➢ All the peptide bonds are cleaved, yielding the aminoacyl hydrazides except the Cterminal residue. ➢ C terminal residue is released as the free amino acid. ➢ Can be identified by chromatography. Chromatography to pully identify str op A A . . Step 2. Cleavage of the disulfide bonds ➢ Cleave the disulfide bonds between Cys residues. ➢ To separate the disulfide bond linked polypeptides (inter chain). ➢ To prevent the native protein conformation that is stabilized by disulfide bonds (Intra chain). ➢ Disulfide bond locations will be established in the final step of the sequencing analysis. ➢ Cleavage reactions are best carried out denaturation conditions (SDS). Method A. Oxidation with performic acid. --- Convert Cys into cysteic acids. ↳ ----Oxidize Met residues. ↓ Cyanogen Bromide cleaves the Bioanalytical Chemistry (Manz et al., 2015) Biochemistry (Voet, 2004) there was she wo that only reacts Met - . side of met resides Step 2. Disulfide-bond cleavage ➢ Disulfide bonds are most often cleaved by reductive treatment with 2mercaptoethanol or dithiothreitol (DTT). ➢ The resulting thiol groups (-SH) are alkylated to prevent the reformation of the disulfide bonds. Biochemistry (Nelson and Cox, 2012) Step 3. Separation, purification, and characterization of the subunits ➢ Each subunits have to be separated and purified. Separation: ➢ Ion exchange chromatography ➢ Gel filtration chromatography. ➢ RP-HPLC Determine the subunit molecular weight: ➢ Each residue is around 110 Da. ➢ Gel filtration or SDS-PAGE ➢ Mass spectroscopy (accurate and fast) Biochemistry (Voet, 2004) https://www.bio-rad.com Table Standards for determining molecular weight. Step 4. Determination of amino acid composition ➢ The amino acid composition is a characteristic parameter for each protein. ➢ Can compare to databases. ➢ The composition is determined by complete hydrolysis followed by the quantitative analysis. ➢ Chemical (acid or base) method ➢ Enzymatic method Biochemistry (Voet, 2004) Bioanalytical Chemistry (Manz et al., 2015) Acid-catalyzed hydrolysis ➢ ➢ ➢ ➢ ➢ ➢ ➢ Treated with 6M HCl, under vacuum condition to prevent oxidation. Heated at 100-120°C for 10-100 h. Longer reaction times to release aliphatic amino acids (Val, Leu, Ile). Ser, Thr and Tyr are partially degraded. Need correction factors. Destroy Trp residues. Gln and Asn are converted to Glu and Asp. Asx(=Asp+Asn), Glx(=Glu+Gln) Base-catalyzed hydrolysis ➢ ➢ ➢ ➢ Treated with 4 M NaOH at 100°C for 4 to 8 h. Cause decomposition of Cys, Ser, Thr and Arg Partially deaminates and racemizes amino acids. Mainly used for Trp content. Step 4. Determination of amino acid composition ➢ Complete enzymatic hydrolysis of a polypeptide requires mixtures of peptidases. ➢ Individual peptidases do not cleave all peptide bonds. ➢ The amount of enzyme loading is limited to 1%. ➢ Often used to determine the Trp, Asn and Gln. ➢ Amino acid quantification can be determined in a reversed-phase HPLC. ➢ Derivatized by treating with ophthalaldehyde (OPA) and 2mercaptoethanol. Biochemistry (Voet, 2004) https://www.thermofisher.com; http://www.scielo.br Step 5. Specific peptide cleavage reactions ➢ Polypeptides that are longer than 40-100 residues cannot be directly sequenced. ➢ Above 50 residues, results become unreliable due to incomplete reactions and the accumulations of impurities. ➢ Samples must be cleaved into small fragments prior to sequencing. ➢ Often divide into two aliquots, and fragmented by different agents leading to different sets of fragments. ➢ After sequencing, the fragments can be compared and ordered due to partial overlap. Biochemistry (Nelson and Cox, 2012) Table Some common methods for fragmentation. 7 6 Trypsin specifically cleaves peptide bonds after positively charged residues ➢ Trypsin has good specificity and cleaved peptide bonds on the carboxyl terminus of the positively charged residues. ➢ C side of Arg and Lys residues, if next residue is not Pro. ➢ The cleavage sites of trypsin can be removed or added. ➢ If positive charge in Arg or Lys is eliminated, trypsin no longer cuts the peptide at this point. ➢ Additional cleaving sites can be generated by introducing a positive charge into side chains. Biochemistry (Voet, 2004) Bioanalytical Chemistry (Manz et al., 2015) 7 7 Cyanogen bromide specifically cleaves peptide bonds after Met residues ➢ Some chemical reagents promote peptide bond cleavage at specific residues. Enzymes ➢ Cyanogen bromide (CNBr) make specific cleavage on the C-side of Met residues. ➢ Form peptidyl homoserine lactone. ➢ Perform in the acidic solvent (0.1 M HCl). 1 Fragmentation is insufficient ❖ Depends on the size of the fragmented peptide, a second run of fragmentation may be needed. ❖ The fragments should be separated and purified for subsequent sequence determination. Fragments can beporified. Biochemistry (Voet, 2004) Bioanalytical Chemistry (Manz et al., 2015) Step 6. Sequence determination ➢ The peptide fragment is sequenced through repeated cycles of the Edman degradation. ➢ An automated device, known as sequenator, was first developed by Edman and Goeffrey Begg. ➢ Peptide is adsorbed on to a PVDF membrane or glass fiber paper. ➢ Reagents are injected by a pumping system ➢ The thiazoline-amino acids are automatically removed, converting to PTH-amino acids. ➢ The PTH-amino acids are identified via HPLC. https://www.protein.iastate.edu/nseque ncePPSQ.html Biochemistry (Voet, 2004) http://tools.thermofisher.com; https://en.wikipedia.org Step 7. Ordering the peptide fragments ➢To elucidate the order that they are connected in the original polypeptide. ➢By comparing the amino acid sequences of one set of peptide fragments with those of a second set whose specific cleavage sites overlap those of the first set. ➢The overlapped peptide segments must be of sufficient length to identify each cleavage site uniquely. Biochemistry (Voet, 2004) Biochemistry (Hames and Hooper, 2005) Step 8. Assignment of disulfide bond positions ➢ Determine the positions of disulfide bonds. ➢ Using the same separating conditions in both dimensions for 2D electrophoresis method. ➢ Native protein is cleaved into fragments. ➢ After the separation in the first dimension, the matrix is exposed to performic acid, which cleaves all disulfide bonds. ➢ After the separation in the second dimension, fragments with disulfide bond show two spots for the two fragments. ➢ The fragments on gel can be isolated and sequenced. ➢ Other methods: HPLC, NMR, Xray crystallography, MS Bioanalytical Chemistry (Manz et al., 2015) Rinalducci et al. (2008) Fig. Separation of fragmented peptides using 2D-SDS–PAGE. Step 8. Assignment of disulfide bond positions ➢ Determine the positions of disulfide bonds. ➢ Using the same separating conditions in both dimensions for 2D electrophoresis method. ➢ Native protein is cleaved into fragments. ➢ After the separation in the first dimension, the matrix is exposed to performic acid, which cleaves all disulfide bonds. ➢ After the separation in the second dimension, fragments with disulfide bond show two spots for the two fragments. ➢ The fragments on gel can be isolated and sequenced. ➢ Other methods: HPLC, NMR, Xray crystallography, MS Bioanalytical Chemistry (Manz et al., 2015) Rinalducci et al. (2008) Fig. Separation of fragmented peptides using 2D-SDS–PAGE.