Protein Extraction: Cell Disruption Techniques

Protein Preparation

• The initial step in protein analysis involves disrupting the cell.
• Two primary methods to disrupt cells are gentle cell disruption and mechanical rupture.

Gentle Cell Disruption

• Gentle cell disruption is suitable for cells that lyse easily, for example red blood cells and tissue culture cells.
• Osmotic Lysis is a popular method where cells swell then burst in hypotonic solutions, releasing their contents.
• Freeze-Thaw Lysis involves lysing cells via quick freezing (using liquid nitrogen) and thawing cycles.
• Detergent Lysis can solubilize cellular membranes using detergent-containing solutions.
• Enzymatic Lysis uses enzymes to digest cell walls in isosmotic solutions.
• Cellulase and Pectinase are used for plant cells, lyticase for yeast cells, and lysozyme for bacterial cells to carry out enzymatic lysis of the respective cell.

Mechanical Rupture

• Mechanical rupture is used to disrupt biological material with tough cell walls and many tissue types.
• Sonication disrupts cell suspensions (cooled to prevent overheating) using shear forces from ultrasonic waves.
• French Press lyses cells with shear forces by forcing cell suspension through small opening at high pressure.
• Mortar and Pestle can break cells of solid tissues and microorganisms and the sample is typically filled with liquid nitrogen to grind tissues or cells into a fine powder.
• Mechanical Homogenization disrupts soft, solid tissues via handheld devices, blenders, or motorized homogenizers.
• Glass Bead Homogenization employs small glass beads to aid cell disruption, mainly for microbial cells.

Protein Quantitation

• Protein quantitation is detected by spectrophotometry, requiring protein standards.
• UV-visible spectroscopy is the foremost cost- and time-efficient method for determining total protein concentration.
• Three types of Spectrophotometric assays are UV absorbance and Dye-binding (colorimetric and fluorescent-based) methods.

Criteria for Assay Selection

• When selecting an assay, sample volume, impacts sensitivity and dynamic range, specifically fluorescence-based assays provide the best results.
• Microplate assays use lower sample volumes and improve sensitivity, typically up to 10-fold higher versus cuvette-based assays.
• For limited samples, a non-destructive method like UV spectroscopy is more appropriate to maximize sample recovery.
• For multiple samples,rapid one-step assays are best for throughput.
• Absorbance-based dye-binding assays have enhanced repeatability and robustness compared to fluorescent assays.
• Chemical modifications (glycosylation or PEGylation, etc.) can interfere with assays.
• Protein solubility often affects response and protein aggression affects expected results.

UV Spectroscopy at 280nm

• Amino acid residues with aromatic rings absorb light at 280nm.
• Follows the Beer-Lambert law, where absorbance is proportional to concentration and path length.
• The method depends on aromatic amino acids, mainly tryptophan and tyrosine in proteins.
• It effectively detects proteins ranging from 20 to 3000 μg.
• It is useful for detecting proteins eluting from chromatography columns without protein loss.
• Advantages include fast, easy automation, and reasonable sensitivity without destroying protein.
• Disadvantage is that some buffers, other reagents, and DNA/RNA interfere with absorbance readings.

Issues with UV Spectroscopy at 280nm

• DNA and RNA exhibit maximum absorbance at 260 nm but still absorb at 280 nm.
• DNA and RNA have tenfold higher absorbance values at 280 nm compared to equivalent protein concentrations.
• Nucleic acids in samples can lead to an inaccurate protein concentration reading using absorbance at 280 nm.

Resolving Nucleic Acid Contamination

• Absorbance measures at 260 nm and 280nm help resolve protein concentration with nucleic acid contamination.

Alternative Methods

• Amino acid analysis (AAA) is effective for identifying and quantifying proteins which involves hydrolyzing peptide bonds to free individual amino acids.

UV Spectroscopy at 205nm

• This assay can quantitate protein solutions at concentration of 1–100 μg/mL.
• It is based on absorbance by the peptide bond.
• Quantitation of proteins by peptide bond absorption at 205nm is more universally applicable than A280.
• The absorptivity for a given protein at 205nm is several-fold greater than at 280 nm, quantifying lower concentrations of protein.

Spectrofluorometric Measurement

• Measures intrinsic fluorescence based on aromatic amino acids like tryptophan, tyrosine, and phenylalanine to determine protein concentration.

Colorimetric Protein Assays

• Colorimetric protein assays need a protein standard to estimate concentration.
• The best standard is the exact same protein in a matched matrix/solution, assigned using amino acid or gravimetric analysis.
• Bovine Serum Albumin (BSA) may give misleading results in sequence-sensitive assays.
• Other options are bovine gamma globulins or immunoglobulins (used for antibody quantitation).

Colorimetric Assay Techniques

• Several colorimetric protein assay techniques are biuret reaction, Lowry, Bradford, and BCA methods in addition to standards and assay validation.

Biuret Reaction

• The biuret reaction involves complex formation between cupric ions and proteins.

Reaction Mechanism for Biuret reaction

• Copper sulfate is added to a protein solution with a strong alkaline solution.
• Complex formation between cupric ions and peptide bonds produces a purplish-violet color.
• The reaction remains independent of protein composition but might be influenced by protein purity and association state.

Reverse Biuret Method

• A modified "reverse biuret method" increases sensitivity by utilizing excess cupric ions, which are reduced by ascorbic acid cuprous ions.

Cu+-bathocuproine

• These then form a complex with bathocuproine, where the amount of Cu+-bathocuproine complex is inversely proportional to protein concentration.

Lowry Method

• The Lowry protein assay is an enhancement of the biuret reaction, by incorporating additional steps and reagents to increase detection sensitivity.

Reaction Mechanism for Lowry Method

• Copper interacts with four nitrogen atoms of peptides that forms a cuprous complex.
• Folin-Ciocalteu reagent interacts with cuprous ions and side chains of tyrosine, tryptophan, and cysteine.
• This produces a blue-green color detectable between 650 nm and 750 nm.

Sensitivity and Detection Range Of Lowry Method

• Detects protein concentrations from 5–100 μg.
• Almost 100-fold more sensitive than direct absorbance measurement at 280 nm.

Limitations and Interferences of Lowry Method

• The protein composition of the sample might not match calibration standards, introduces potential error.
• Is susceptible interferences from substances such as K+, Mg2+, NH₄⁺, EDTA, Tris-HCl, carbohydrates, and reducing agents
• Folin reagent only remains reactive for a short time after addition
• Destructive assay since, once reacted, the protein sample cannot be used for other assays

Bradford Method

• The Bradford protein assay employs coomassie brilliant blue G-250 dye to bind proteins, this forms the dye-protein complex with increased molar absorbance, which allows for protein concentration determination.

Bradford Method Reaction Mechanism

• Coomassie brilliant blue G-250 is protonated and shows reddish-brown color with an absorbance maximum of 465 nm at acidic pH.
• Under acidic conditions, the dye interacts with arginine and to a lesser extent, lysine, histidine, tyrosine, tryptophan, & phenylalanine residues.
• This reaction produces a blue color with an absorbance maximum at 595 nm, the range includes (575–615 nm), this allows for detection of 0.2–20 μg of protein

Advantages of Bradford Method

• It is fast, simple, and highly sensitive compared to other protein determination methods.
• The dye is stable for long periods.
• The assay has reduced reagent volumes and it is adapted for a 96-well plate format.

Limitations and Considerations for Bradford Method

• Dye binding depends on basic amino acid content, making standard selection crucial for accuracy.
• The Coomassie dye stains cuvettes, and the stain cleans with dilute SDS solution.
• Protein-to-protein variability can be minimized by decreasing acidity with NaOH, or by including Triton X-100 or SDS.
• Concentrated protein solutions might precipitate upon contact with the dye reagent, requiring dilution for measurements.
• The assay is destructive to proteins because after they react they aren't reused for other assays.

BCA Method

• The BCA protein assay is the reaction between bicinchoninic acid and cuprous ions produced by the biuret reaction under alkaline conditions.

BCA Method Reaction Mechanism

• Sodium salt of bicinchoninic acid reacts with cuprous ions to generate a deep blue complex.
• The absorbance is read at 562nm.
• The detection range is 0.2–50 μg of protein.

Advantage of BCA Method

• The BCA reagent is stable under alkaline conditions and can be included in the biuret alkaline copper solution.
• Detergents are more compatible compared to Lowry and Bradford assays.
• It has less protein-to-protein variability compared to the Bradford assay.
• Protein composition variability is minimized by conducting the reaction at 60°C.
• Performed in a 96-well plate with reduced reagent volumes.

Limitations and Interferences BCA Method

• Reducing agents (which reduce cupric to cuprous ions) interfere with the assay.
• Chelating agents (EDTA) interfere because it binds to the copper.
• Hydrogen peroxide (H₂O₂) and phospholipids interfere with phospholipids, producing artificially high values due to chromophore formation at 562nm.
• Destructive assay, after the reaction with copper, the protein is cannot be use for other assays.

Fluorescent Dye-Based Assays

• Fluorescent dye assays uses Microplate detection and Cuvette detection methods.

Microplate Detection Method

• Amine-labeling derivatization uses varied fluorescent probes to determine amino acid mixtures in amino acid analysis.
• Used for protein/peptide quantification, particularly for proteins with a free N-terminus or lysine (accessible to the dye).

Microplate Reaction Mechanism

• After reaction with amines, an elevated level of fluorescence can be detected with a linear response, this occurs within a dynamic range of 0.05–25 μg of protein.
• Fluorescent dyes used in microplate protein quantification includes o-Phthalaldehyde (OPA), Fluorescamine (reacts with amine functional groups), and 3-(4-Carboxybenzoyl)quinoline-2-carboxyaldehyde (CBQCA) (requires a thiol such as 2-mercaptoethanol or cyanide).

Cuvette Detection Method

• In a cuvette format, when OPA reacts with primary amino acids, except cysteine, then a highly fluorescent adduct is formed.

Cuvette Reaction Mechanism

• OPA reacts with 4-amino-1-butanol and 4-aminobutane-1,3-diol, produced by oxidation of proline and 4-hydroxyproline, in conjunction with chloramine-T plus sodium borohydride at 60°C.
• OPA also reacts with S-carboxymethyl-cysteine, formed from cysteine and iodoacetic acid at 25°C.
• OPA derivatives are monitored at excitation and emission wavelengths of 340 nm and 455 nm.
• The detection limit for amino acids is 50 fmol.

Limitations of Fluorescent Dyes

• Dyes offer better sensitivity and dynamic range than absorbance-based protein quantitation assays.
• OPA is often preferred over fluorescamine, due to its improved stability and solubility.

Molecular Separation via Column Chromatography

• Cell extracts are further purified via various biochemical techniques through various methods of biochemical techniques.
• Column chromatography uses a glass or plastic tube filled with a resin matrix separation.

Process of Column Chromatography

• The protein sample is applied to the top of the column.
• A buffer solution continuously flows through the column.
• Proteins migrate through a column at different rates depending on: the nature of the matrix and the physical and chemical properties of the proteins.
• The targeted protein is separated from the other proteins in the sample.
• The method purifies proteins based on specific interactions with the chromatography matrix.

Size Exclusion Chromatography

• Size exclusion separates proteins by size and shape via a gel filtration column.
• The column matrix, often called resin, is made of inert material that contains tiny pores, and is similar to a whiffle ball.

Mechanism of Separation in Size Exclusion Chromatography

• Small molecules that are smaller than cut-off then enter the pores of the matrix, but after that they get trapped in the resin and progress slowly.
• The molecule is excluded from the pore as the large molecules cannot fit into the pore.
• Big molecules move very fast and they make their way through the column by travelling between the resin beads, shorter distance.

Elution order in size exclusion chromatography

• Large molecules emerge first from the column.
• Small molecules will emerge last as they stay behind longer inside the resin pores.
• Effectively separates proteins based on molecular size, useful for protein purification and molecular weight determination.

Ion-Exchange Chromatography

• Ion-exchange chromatography separates proteins depending on surface charge by employing resin that contains positively or negatively charged chemical groups.

Types of Ion-Exchange Resins

• Anion-Exchange resins: positive groups attract negatively charged proteins.
• Cation-Exchange resins: negative groups attract positively charged proteins.

Mechanics of Separation in Ion-Exchange Chromatography

• In low-salt solutions, proteins with negative surface charge bind strongly to positively charged anion-exchange columns.
• A protein that has a positive charge will bind to negativity charged column called cation-exchange columns.
• Higher buffer concentrations causes the resin to strongly bind onto the bound proteins, causing them to move out or elute from the column.
• This helps separation based on charge and adjusting p.H.

Affinity Chromatography

• Separates proteins depending on it, binding affinity.
• A ligand specific to the protein of interest is immobilized on resin.
• The Target protein binds to the ligand, isolating it from the mixture.
• Elution comes about because of High concentrations of free ligand.
• Antibody-coated resin binds to its target protein.
• High specificity causes it to purify it in one step.

Hydrophobic Interaction Chromatography

• Principle: Separates proteins based on hydrophobicity.
• A hydrophobic group has to be used , then the phenyl group is covalently attached to the column matrix.
• High-salt buffer enhancers promotes protein binding to the resin.
• Elution is possible if the proteins are eluted by Lowering salt concentration and Adding solvents such as polyethylene glycol.
• It can Purify proteins without causing damage or denaturation of sample.

Elution Order in HIC

• Most Hydrophobic Proteins Elute First then with each level moving a step below, a high interaction with the resin can occur.
• Highly Hydrophobic Proteins would have a tough time, and would use substantial reduction in salt in order to come out

Gel-Free Proteomics

• Relies on the technique of using liquid chromatography with a high speed.
• Used in proteomics as well as high resolution studies
• Using liquid chromatography at a high speed allows the detection of Molecules at a speed of 1.0 Nm
• Allows study in research and genomics.

Types of Liquid Chromatography in Proteomics are:

• Affinity type in chromatography
• Gel Permeation is chromatography,
• Ligand exchange is in chromatography.
• Capillary

Reversed-Phase HPLC (RP-HPLC) -Chromatography

• It allows a very wide number of applications and a lot of stages on the way, that is why it is very popular to use..

Stationary Phase in Chromatography

• Uses a non-phase alky hydocarbon, that binds onto silica.
• A popular step used is the C18 when doing the matrix test
• Long chains is key when it comes to high hydrobotic.
• Lower can result in lower level activity as well in order to have the chain

Polar

• Mobile is polar and allows for Less polar

Affinity HPLC

• Relies on the use of stationary that allows you to hold then reverse the system.
• works similar to affinity chromo.
• You can now purity the set of molecule.

Gel type HPLC

• Seperates molecules that may have size differential , uses seperation.
• The larger samples come first when analyzing You need a large and porous solid.
• Protein isolation uses Sep harose in order to use it in chromatography