Techniques in Protein Quantification & Analysis PDF

TECHNIQUES IN PROTEIN QUANTIFICATION & ANALYSIS Molecular Biology and Diagnostics PROTEIN QUANTITATION Proteins – also known as polypeptides, are organic The method depends on the relative number of compounds made up of amino acids....

TECHNIQUES IN PROTEIN QUANTIFICATION & ANALYSIS Molecular Biology and Diagnostics PROTEIN QUANTITATION Proteins – also known as polypeptides, are organic The method depends on the relative number of compounds made up of amino acids. aromatic rings, primarily the tryptophan and the They are large, complex molecules that play many tyrosine residues present in a protein. It is useful for critical roles in the body. detection of proteins eluting the chromatography They do most of the work in cells and required for columns as there is no loss of proteins. So the the structure, function, and regulation of the body possibility of nucleic acid contamination should also tissues and organs. be considered and compensation can be made. They also assist with the formation of new molecules by reading the genetic information Advantages of using absorbance at 280 nm: stored in DNA molecules. Fast, easily automated, reasonably sensitive & do not destroy protein Spectrophotometric assays of protein in solution → Disadvantages of using absorbance at 280 nm: do not require either enzymatic/chemical digestion or Many buffers and other reagents can interfere with separation of the mixture prior to analysis A280 spectrophotometric measurements The A280 measurement is also interfered with by Three major types of spectrophotometric assays: the absorbance from DNA and RNA (have tenfold 1. UV absorbance methods higher absorbance values at 280nm compared to 2. Dye-binding assays using colorimetric- based the equivalent concentration of protein) detection 3. Dye-binding assays using fluorescent- based To resolve the protein concentration in such detection samples, measure the absorbance at 260 nm and 280nm and calculate the protein concentration as Although these assays can be run at a high throughput, follows: they require an appropriate protein standard or constituents amino acid sequence information to make an estimate of the concentration. The choice of method *This estimation of protein concentration is valid up to used to determine the concentration of the protein or 20% (w/v) nucleic acid or an A280/A260 ratio less than peptide in a solution depends on many factors. 0.6 Detecting Proteins With Absorbance at 205nm 1. UV SPECTROSCOPY METHOD: Determination of protein concentration by Detecting Proteins With Absorbance at 280nm measurement of absorbance at 205nm (A205) is At 280nm, amino acid residues with aromatic based on absorbance by the peptide bond rings in a protein absorb light This assay can be used to quantitate protein The method follows the Beer-Lambert law in solutions with concentrations of 1–100μg/mL which absorbance is proportional to protein concentration and path length The absorptivity for a given protein at 205nm is The amino acids tryptophan (λmax 279.8nm) and several-fold greater than that at 280 nm tyrosine (λmax 274.6 nm) contribute most to the Absorbance at 205 nm can be used to quantitate absorbance at this wavelength, while dilute solutions, or for short path length phenylalanine (λmax 279 nm) makes a minor applications contribution The disadvantage of this method is that some The method can be used to detect protein in the buffers and other components also absorb at 20–3000μg range 205nm  TECHNIQUES IN PROTEIN QUANTIFICATION & ANALYSIS Molecular Biology and Diagnostics 2. Lowry Method based on the biuret reaction with additional steps and reagents to increase the sensitivity of detection Lowry adds phosphomolybdic /phosphotungstic acid also known as Folin- Ciocalteu reagent = (+) blue-green color @ 650nm-750nm The protein detection range is 5–100μg 3. Bradford Method uses the binding of Coomassie brilliant blue Spectrofluorometric Measurement: G-250 dye to proteins Detecting Proteins by Intrinsic Fluorescence The Coomassie brilliant blue G-250 dye is Emission protonated and is reddish/brown with an Protein concentration can also be determined by absorbance maximum of 465 nm at acidic Ph measuring the intrinsic fluorescence based on Under acidic conditions, the dye reacts fluorescence emission by the aromatic amino primarily with arginine and to a lesser extent acids tryptophan, tyrosine, and phenylalanine with lysine, histidine, tyrosine, tryptophan, This assay can be used to quantitate protein and phenylalanine residues in proteins (+) solutions with concentrations of 5–50μg/mL blue color with an absorbance maximum at Normally, the excitation wavelength is set to 280 595nm nm and the emission wavelength is between 320 0.2–20 μg of protein can be detected and 350 nm 4. Bicinchoninic Acid Assay (BCA) Method 2. COLORIMETRIC PROTEIN ASSAY TECHNIQUES based on the fact that the sodium salt of bicinchoninic acid reacts with the cuprous ion All colorimetric protein assays require protein generated by the biuret reaction under alkaline standard to estimate the concentration of a sample conditions. The bicinchoninic acid cuprous complex (+) Commercially available standards : deep blue color st 562nm 1. Bovine serum albumin (BSA) → Detection range is 0.2–50μg 2. Bovine gamma globulins 3. Immunoglobulins 3. FLUORESCENT DYE-BASED ASSAYS COLORIMETRIC PROTEIN ASSAY TECHNIQUES 1. Microplate Detection Method 1. Biuret reaction This technique can be used to quantitate based on the complex formation of cupric ions proteins and peptides containing either lysine with proteins or a free N terminus In this reaction, copper sulfate is added to a Upon reaction with amines, the dyes display a protein solution in strong alkaline solution = large increase in fluorescence that (+) purplish-violet color The range of this method is 0.05–25 μg  TECHNIQUES IN PROTEIN QUANTIFICATION & ANALYSIS Molecular Biology and Diagnostics Three dyes used to quantitate proteins, or amino acids 2. Size Exclusion Chromatography in a microplate format: This chromatography can separate proteins 1. Ophthalaldehyde (OPA)* based on the size and shape on a gel filtration 2. Fluorescamine column 3. 3-(4 carboxybenzoyl)quinoline-2-carboxyaldehyde The column matrix also known as resin (CBQCA)* consists of microscopic beads of inert material *require the addition of a thiol (2-mercaptoethanol) or Larger molecules can only pass through the cyanide spaces between resin beads 2. Cuvette Detection Method OPA reacts with primary amino acid (except cysteine) in the presence of 2 mercaptoethanol or 3-mercaptopropionic acid to form a highly fluorescent adduct OPA also reacts with 4-amino-1-butanol and 4-aminobutane-1,3-diol produced from oxidation of proline and 4-hydroxyproline Fluorescence of OPA derivatives is monitored at excitation and emission wavelengths of 340 nm and 455 nm Detection limits are 50fmol for amino acid MOLECULAR SEPARATION OF PROTEIN MOLECULES THROUGH COLUMN CHROMATOGRAPHY The most common method for purifying proteins from other protein molecules this method uses a glass or plastic tube filled with resin that can separate proteins based on their 2. Ion-Exchange Chromatography physical properties as they flow through the Uses a resin to separate proteins according to column their surface charges This type of column contains a resin bearing either positively or negatively charged chemical groups  TECHNIQUES IN PROTEIN QUANTIFICATION & ANALYSIS Molecular Biology and Diagnostics Each sodium dodecyl sulfate contributes two negative charges SDS is also a detergent that disrupts protein tertiary structure SDS has two advantages: a) It coats all the polypeptides with negative charges b) It masks the natural charges of the subun Staining with the organic dye like Coomassie blue Coomassie Brilliant Blue is the most frequently used. 3. Affinity Chromatography Uses the principle that the protein binds to a After reduction and denaturation by SDS: molecule for which it has specific affinity Proteins migrate in the gel according to their molecule can be immobilized through molecular mass covalent attachment to the resin in a column The electrophoretic mobility of proteins upon SDS- The power of affinity chromatography lies in PAGE is inversely proportional to the logarithm of the specificity of binding between the affinity the protein’s molecular weight reagent on the resin and the molecule to be SDS-PAGE is often used to determine the purified molecular weight of proteins Gel Staining After Electrophoresis: Colorimetric Staining Methods MOLECULAR SEPARATION THROUGH These can detect as low as 10-ng and 1-ng protein ELECTROPHORESUS bands 1. Sodium Dodecyl Sulfate-Polyacrylamide Gel Three high-sensitivity colorimetric staining methods Electrophoresis (SDS-PAGE) can be used either directly after electrophoresis: The hydrophobic tail of SDS interacts strongly 1. Coomassie blue staining – anionic with polypeptide chains triphenylmethane dye The number of SDS molecules bound by a Modifications: Coomassie R-250 (Red tint) polypeptide is proportional to the length of and Coomassie G-250 (Green tint) the polypeptide In the presence of the acidic medium these dyes stick to the amino acids of the proteins by electrostatic and hydrophobic interaction.  TECHNIQUES IN PROTEIN QUANTIFICATION & ANALYSIS Molecular Biology and Diagnostics 2. Zinc-reverse staining – also called as negative of microorganisms, cells, and heterogeneous staining which uses imidazole and zinc salts for tissues. protein detection in electrophoresis gels It is based on the precipitation of the zinc Gel-based proteomics: imidazole in the gel zinc imidazole except in the 1. Two-dimensional gel electrophoresis (2DE) gel zones where proteins are located. When zinc 2. 2DE fluorescence differential imaging gel reverse staining is applied on a Kumasi blue electrophoresis (DIGE) stain gel previously undetected bands can be spotted. Gel-free proteomics not seen in the Kumasi blue staining can be 1. Liquid chromatography (LC) seen using the zinc reverse staining This 2. Capillary electrophoresis (CE) technique is rapid, simple, cheap and reproducible and is and it is compatible with Gel-based Proteomics Microsoft analysis Two-Dimensional Gel Electrophoresis In 2DE, the complex protein samples are 3. Silver staining – based on the binding of silver separated in two dimensions according to their: ions to the proteins followed by reduction to free 1. Net charge at different Ph silver, sensitization, and enhancement 2. Molecular weights determined by SDS- detect the proteins with silver because of the PAGE sensitivity below 1 nanogram per spot. Protein migration in a perpendicular It is cheaper direction provides a spot map of the used as a second staining and essential to fix proteins distributed in 2DE gel the proteins in the gel acidic alcohol prior to Resolving power= >10,000 spots initial Coomassie blue staining. corresponding to >1000 proteins. Fluorescence Staining Method Considered as endpoint method thus the results are highly reproducible Require a fluorescence imager for visualization and that they are significantly more expensive than classical colorimetric dyes e.g Nile Red, ruthenium(II) tris(bathophenantroline disulfonate) (RuBPS), SyPro (orange, red, tangerine, ruby, deep purple), Epicocconone, CyDyes GEL-BASED PROTEOMICS: PROTEIN SEPARATION most popular and well-established technique for First Dimension Electrophoresis: global protein separation and quantification. 1. Conventional Isoelectric Focusing The field that uses techniques to resolve the complex protein samples are separated thousands of polypeptides is called proteomics according to their net charge at different pH 2DE gel coupled with MS → most reproducible and the mixture of proteins is electrophoresed effective technology to separate overall proteins through a narrow tube gel containing molecules called ampholytes  TECHNIQUES IN PROTEIN QUANTIFICATION & ANALYSIS Molecular Biology and Diagnostics Isoelectric point (pI) The pH at which the protein has no net charge Protein is no longer drawn toward the anode or the cathode → STOPS Modification of 2DE: Two-Dimensional Difference Gel Electrophoresis Differential Imaging Gel Electrophoresis (DIGE) Uses size- and charge-matched cyanine dyes (CyDye, DIGE, Fluors) with different excitation and 2. Immobilized pH Gradient (IPG) emission wavelengths as protein labels for IPG is an integrated part of a polyacrylamide different samples gel matrix fixed on a plastic strip Using multiplexed fluorescent dyes (e.g., Cy2, Cy3, and Cy5) to covalently label protein samples 3. Nonequilibrium pH Gel Electrophoresis 2D-DIGE can run more than one sample NEPHGE technique can resolve proteins with (maximum 3 samples) on a single gel at once to basic to extremely high pI (7.0–11.0) that address the issue of gel-to-gel variability cannot be separated by traditional method SAMPLE BUFFER CONSTITUENTS FOR 2DE 1. Chaotropic Agents (8-9M Urea) - It effectively disrupts noncovalent and ionic bonds between amino acid residues\ 2. Detergents - Hydrophobic interactions within a polypeptide chain or between proteins in protein complexes can be disrupted in the presence of detergents and it can also increase solubility, especially of membrane proteins 3. Reducing Agents (DTT)- cleave disulfide bond cross-links within and between protein subunits, Second Dimension SDS-PAGE thereby promoting protein unfolding and The second step of 2DE separates proteins based maintaining proteins in their fully reduced states on their molecular weight using a vertical 4. Carrier Ampholytes - They can reduce protein- electrophoretic device with either Laemmli buffer matrix hydrophobic interactions and usually or Tris-Tricine buffer included at a concentration of 0.5%–2% (v/v) in The proteins are resolved according to their sizes sample solutions for IPG strip focusing by SDS-PAGE  TECHNIQUES IN PROTEIN QUANTIFICATION & ANALYSIS Molecular Biology and Diagnostics GEL FREE PROTEOMICS TECHNIQUES: LIQUID CHROMATOGRAPHY CAPILLARY ELECTROPHORESIS Useful in proteomics and genome research typical gel-free technique, with the advantage of because it can detect molecules at the nanomolar superior separation efficiency, small sample level consumption, short analysis time, and automatability Types of Liquid chromatography a) Reversed-phase High performance liquid Protein separation in CE is based on a variety of chromatography (HPLC) properties: b) Affinity HPLC a) Molecular mass (SDS-capillary gel c) Gel permeation HPLC electrophoresis [CGE]) d) Ligand-exchange HPLC a capillary is filled with gel or viscous solution e) Capillary HPLC (to form molecular sieve) and the electroosmotic flow (EOF) is usually 1. Reversed-Phase HPLC suppressed Most popular mode of chromatography b) Isoelectric point (capillary isoelectric focusing because of its wide range of applications and [CIEF]) the availability of various mobile and stationary c) Electrophoretic mobility (capillary zone phases electrophoresis [CZE]) sample is introduced into a buffer-filled 2. Affinity HPLC capillary either electrokinetically (with low Chromatographic method capable of voltage) or hydrodynamically (with pressure or separating biochemical mixtures of highly suction specific nature MICROCHIP ELECTROPHORESIS 3. Gel-Permeation HPLC Miniaturized form of conventional CE Works on the principle of sizes of the Could integrate injection, separation, and compounds, which is the same as the size- detection on a single microchip with typical exclusion chromatography channel lengths of 5 cm to 15 cm It is a method of choice for separation of Compared to conventional CE the advantage to biomolecules such as peptides, proteins, and use ME include shorter analysis time, less enzymes sample/reagent consumption, and less waste generation 4. Ligand-Exchange HPLC ME enables fast separation of protein, peptide, Advanced version of reversed-phase-HPLC and single-cell analysis, which are normally where the reversed-phase column is replaced present in small amounts in complex matrices. by an ion-exchange column It has been used widely for the analysis of all inorganic and organic ionic species 5. Capillary Electrochromatography A hybrid technique of HPLC and CE It combines the high peak efficiency that is characteristic of electrically driven separations with high separation selectivity  TECHNIQUES IN PROTEIN QUANTIFICATION & ANALYSIS Molecular Biology and Diagnostics Uses pre-stained molecular-weight standards such as cytochrome C (11,700 d) to myosin (205,000 d) Gel pre-treatment (20% glycerol in 50 mM Tris- HCl, pH 7.4) can renature proteins before transfer After electrophoresis, proteins are blotted to nitrocellulose membrane by capillary or electrophoretic transfer Nitrocellulose has a high affinity for proteins and is easily treated with detergent (0.1% Tween 20 in 0.05 M Tris and 0.15 M sodium chloride, pH 7.6) with 5% dry milk to prevent binding of the primary PROTEIN ANALYSIS: MASS SPECTROMETER antibody probe to the membrane itself (blocking) Exploit the difference in the mass-to-charge (m/z) before hybridization ratio of ionized atoms or molecules to separate After incubation with the primary antibody for 12 them from each other to 16 hours, the blot is washed in the same buffer (without dry milk) and incubated with the The basic operation of a mass spectrometer is to: secondary antibody conjugated with enzyme 1. Evaporate and ionize molecules in a vacuum, The blot is washed again to remove excess creating gas-phase ions secondary antibody conjugate, and the 2. Separate the ions in space and/or time based on chemiluminescent or color signal is developed their m/z ratios with the addition of substrate. 3. Measure the quantity of ions with specific m/z Other membrane types used for protein blotting ratios are PVDF and anion (DEAE) or cation (CM) exchange cellulose PROTEIN ANALYSIS: WESTERN BLOT WESTERN BLOT The two most prominent MS methods for protein Western blot protein probes are antibodies analysis are electrospray ionization MS (ESI-MS) (polyclonal or monoclonal) that bind specifically and matrix-assisted laser desorption/ionization- to the immobilized target protein time of flight MS (MALDI-TOF MS) Polyclonal antibodies are comprised of a mixture of immunoglobulins directed at more than one PROTEIN ANALYSIS: WESTERN BLOT epitope on the antigen The immobilized target for a western blot is In western blot technology: protein Polyclonal antibodies can give a more robust Serum, cell lysate, or extract is separated on SDS- signal, especially if the target epitopes are polyacrylamide gels (SDS-PAGE) or isoelectric partially lost during electrophoresis and focusing gels (IEF) transfer Dithiothreitol or 2-mercaptoethanol may also be Monoclonal antibodies are more specific and used to separate proteins into subunits may give less background noise Polyacrylamide concentrations vary from 5% to 20% & 1 to 50 μg of protein is loaded per well Before loading, the sample is treated with denaturant, such as mixing 1:1 with 0.04 M Tris HCl, pH 6.8, 0.1% SDS  TECHNIQUES IN PROTEIN QUANTIFICATION & ANALYSIS Molecular Biology and Diagnostics The protein probes used in western blot applications may be labeled with: 1. 35S 2. Horseradish peroxidase (HRP) or alkaline phosphatase (AP) When exposed to a light or color generating substrate, the enzyme will produce a detectable signal on the membrane or on an autoradiogram ELISA CONFIRMATION Application of the western blot method is confirmation of enzyme-linked immunoassay (ELISA) results for human immunodeficiency virus (HIV) and hepatitis C virus (HCV)