Protein Isolation and Analysis Notes

Isolation and Analysis of Proteins

Introduction

• Test diversity, distribution, and function of proteins.

• Key questions to consider:

  • What is the molecular mass of the protein?

    • Ranges from $6,000$ Da to $1,000,000$ Da.

    • Molecular mass influences the choice of purification method.

  • What are the shape and charge of the protein and its various molecular forms?

    • Most proteins are natively globular.

    • Purification and analysis methods are often tailored to globular proteins.

    • Charge is important when selecting anionic or cationic ion-exchange matrices.

  • Is the protein membrane-bound?

    • Membrane-bound proteins require detergents to stay in solution.

  • What is the copy number of the protein in the cell?

  • What is the impact of the type and physiological state of the cells?

  • Is the protein found in protein complexes?

  • What are the kinetics and thermodynamics of protein complex formation?

  • Is the protein present in more than one cellular compartment?

  • How many molecular forms of the protein are there?

Purification Scheme: Isolation of Intracellular Proteins from Yeast

• The first step is to generate a crude protein extract.

• Cell extracts typically contain $10-30$ mg/ml of total protein.

• This concentration is strain and organism dependent.

• Protocols for yeast cell breakage:

  1. Spheroplasts

  2. Liquid nitrogen

  3. Glass beads

Spheroplasts

• Cells are converted to spheroplasts (Zymolyase treatment) and then lysed by osmotic shock and homogenization.

• This is the gentlest method for breaking yeast cells.

• It is used to prepare extracts with intact complexes.

• Disadvantages: tedious and expensive.

• Critical parameters:

  • Handle spheroplasts gently.

  • Optimize treatment time and concentration of Zymolyase.

Liquid Nitrogen

• Cells are pelleted by centrifugation and frozen in liquid nitrogen.

• Lysis occurs via blender or mortar and pestle in the presence of liquid nitrogen.

• This method is quick and easy, unless there are multiple samples.

• Critical parameters:

  • Ensure most cells are broken for optimal yields (test microscopically).

Glass Beads

• Cells are mechanically broken by vortexing with glass beads.

• This method is very flexible in scale and number of samples.

• It is particularly useful when making extracts from many different small cultures for assays.

• Proteolysis and modification may occur above $4$°C.

• Critical parameters:

  • Use correct bead size for efficient cell breakage.

  • Monitor the degree of breakage.

  • Maintain solution temperature between $0$° and $4$°C.

  • Vortex in bursts, chill on ice.

Other Critical Parameters for Protein Isolation

Buffers

• Select a buffer with a pKa value in the range where your protein is stable.

• Use a buffer with low ionic strength (approximately $50$ mM).

• Salts can influence protein activity.

Salts, Metal Ions, and Chelators

• Avoid Tris-buffers if a metal co-factor is required for protein activity or stability, as Tris binds to metal ions.

Reducing Agents

• Examples: β-mercaptoethanol and DTT reduce oxidation.

• They may cause loss of activity in some cases.

Detergents

• Required to isolate membrane-bound proteins.

• Ionic detergents (e.g., SDS):

  • Separate proteins into monomers and cause denaturation.

• Non-ionic detergents (e.g., Triton X-100, Triton X-114):

  • Cause less denaturation but may form aggregates.

• Zwitterions (e.g., CHAPS and Zwittergent 3-14):

  • Do not stimulate aggregation and are non-denaturing.

Protease Inhibitors

• Include different protease inhibitors at all stages.

• Commercial protease inhibitor mixes, along with PMSF and EDTA, should inactivate almost all proteases.

• Extracts will also contain large amounts of nucleic acid, especially RNA, which may require a nuclease step.

• Cell extracts produced by these procedures will contain approximately $10-30$ mg/ml of total protein (strain and organism dependent).

Isolation of Extracellular Proteins

• Some proteins of interest may be secreted.

• Quantification or analysis may require concentrated supernatant.

• Methods:

  1. Precipitation

  2. Freeze-drying

  3. Ultrafiltration

Precipitation (Salting Out)

• Common first step: ammonium sulfate precipitation.

• Protein solubility depends on the ionic strength of the solution (i.e., salt concentration).

• Proteins differ in their solubility at high ionic strength.

• Add increasing amounts of ammonium sulfate and collect different fractions of precipitated protein by centrifugation.

• Advantages:

  • Ammonium sulfate has very high solubility.

  • Allows preparation of salt solutions with high ionic strength.

  • Inexpensive, even with very large volumes.

  • Non-denaturing.

• Proteins can also be precipitated by trichloroacetic acid (TCA), acetone, ethanol, and a variety of organic solvents.

• While effective, these methods can sometimes lead to denaturation or other issues.

Freeze-Drying

• Lyophilization or cryodesiccation.

• A dehydration process typically used to preserve perishable material.

• Freeze the material, then reduce surrounding pressure and add enough heat to allow frozen water to sublime directly from the solid phase to the gas phase.

• Protein samples are frozen at $-80$°C.

• Add water or buffer to reconstitute.

Ultrafiltration

• A variety of membrane filtration systems exist.

• Hydrostatic pressure forces liquid against a semi-permeable membrane.

• Membranes are specific for molecule size (e.g., $10$ kDa cut-off).

• Suspended solids and solutes of high molecular weight are retained.

• Water and low-molecular weight solutes pass through the membrane.

• A large variety of membranes are commercially available.

• Examples: AmiconTM Ultrafiltration cell, Minitan Ultrafiltration system, Pellicon Ultrafiltration Modules.

• Ultrafiltration can also be used for dialysis or buffer exchange.

Ultrafiltration Modules

AmiconTM

• Gas (N2) pressure is applied directly to the ultrafiltration cell.

• Solutes above the membrane's molecular weight cut-off are retained, while water and solutes below the cut-off pass into the filtrate.

Pellicon & Millipore ultrafiltration systems

• Tangential flow devices use stacked ultrafiltration membranes.

• A peristaltic pump passes fluid from a non-pressurized reservoir across stacked membranes.

• Concentrated material is returned to the reservoir, with filtrate collected separately.

Quantitation of Proteins

• Accurate quantitation at the beginning and end of a series of steps is the only way to evaluate overall yield.

• Indicates steps that may have led to significant protein loss.

• Properties of the protein (e.g., aromatic amino acid content) can suggest a method for analysis.

• Methods:

  • Spectrophotometry

    • Measure absorbance of aromatic amino acids at different wavelengths.

    • Non-destructive and requires very little sample and time.

  • Fluorescence

    • More qualitative, much more sensitive.

  • Colorimetric methods (Bradford and Lowry)

Spectrophotometric Methods

• Absorbance at A280

  • Measures absorbance of UV light by aromatic amino acids (tryptophan and tyrosine) and disulfide-bonded cysteine residues.

  • Absorbance is used to calculate concentration from its known absorptivity at $280$ nm or by comparison with a calibration curve.

  • Quantifies solutions with protein concentrations of $20 – 3,000$ µg/ml.

• Absorbance at A205

  • Based on absorbance by peptide bonds.

  • Quantifies protein solutions with concentrations of $1 - 100$ µg/ml.

Important Parameters for Spectrophotometry

• Use a $1$-cm quartz cuvette.

• Solvent pH and polarity will affect absorbance and fluorescence (e.g., pH effects absorbance of tyrosine residues).

• Reagents containing carbon-carbon or carbon-oxygen double bonds can interfere with the A205 method.

• Calibration curves must be prepared in the same solvent as samples.

• When using a published absorptivity at a given wavelength, the solvent composition of the sample must match that of the published data.

• Stray light can affect the linearity of absorbance versus concentration.

• Absorbance values greater than $1.0$ should not be used (dilute further).

• Nucleic acids have substantial absorbance at $280$ nm and can interfere.

• Estimation of protein concentration is valid up to $20$% (w/v) nucleic acid or A280/A260 ratio < $0.6$.

Protein Concentration @ 280 nm

• Switch on spectrophotometer and activate deuterium lamp.

• Set the wavelength to $280$ nm.

• Wait $10-15$ minutes for lamp to warm up.

• Set up a standard curve using $2$ mg/ml BSA.

• Make a dilution series of $2.0$, $1.5$, $1.0$, $0.5$, $0.2$, and $0.1$ mg/ml.

• Dilute with the same buffer as in the unknown sample.

• Use buffer alone as the blank.

• Measure absorbance at $280$ nm.

• Draw a standard curve of A280 vs protein concentration.

• Measure absorbance of the unknown protein solution at $280$ nm.

• Determine concentration from the standard curve.

• Confirm concentration by using the extinction coefficient of BSA.

Spectrophotometric Methods (2)

• Colorimetric determination via the Bradford method ($595$ nm).

  • Requires a standard curve with known concentrations.

  • $1$ to $10$ µg/ml protein.

• Fluorescence

  • Measure intrinsic fluorescence based on emission by aromatic amino acids (tryptophan, tyrosine, and/or phenylalanine).

  • Concentration is calculated from a calibration curve based on fluorescence emission of standard solutions of pure protein.

  • $5$ to $50$ µg/ml.

Colorimetric Methods

Bradford Method

• Coomassie Brilliant Blue binds to protein.

• Compare absorbance to that of different amounts of standard protein, usually BSA.

• $1$ to $10$ µg protein.

Lowry Method

• Quantitate the color from the reaction of Folin-Ciocalteu phenol reagent with tyrosine residues of protein.

• Compare to a standard curve.

• $1$ to $20$ µg protein.

• Many substances interfere with the Bradford assay, including glycerol, detergents, $2$-mercaptoethanol, acetic acid, ammonium sulfate, Tris, and certain alkaline buffers. Use appropriate controls.

• Many substances also interfere with the Lowry assay. Assess the effect of detergents, denaturants, organic buffers, and/or thiols in the unknown protein solution. Create standard curves based on data taken in both the presence and absence of these compounds.

• Precipitation of protein will eliminate many of these interfering substances.

Colorimetric (Bradford) Assays

• Add $5$, $10$, $15$ and $20$ μl of $0.5$ mg/ml BSA stock solution to 4 different Eppendorff tubes.

• Dilute with $0.15$M NaCl to $100$ μl.

• Use $100$ μl $0.15$M NaCl as blank.

• Add $0.9$ ml Coomassie Blue solution to all tubes and mix well.

• Allow to stand for $5$ minutes at room temperature.

• Measure absorbance at $595$ nm.

• Draw a standard curve of A595 vs protein concentration.

• Measure absorbance of the unknown protein solution at $595$ nm.

• Determine concentration from the standard curve.

• Determinations are usually done in duplicate.

• If the concentration is too high, prepare a dilution or draw a standard curve at higher concentrations (e.g., $0-100$ g/ml).

Methods for Separation and Purification of Proteins

• Methods range from simple precipitation to sophisticated chromatographic and affinity techniques.

• Chromatography

  • Physical separation in which components are distributed between two phases.

  • Stationary phase: immobilized on support particles.

  • Mobile phase: moves in a definite direction (liquid (LC), gas (GC), or supercritical fluid).

  • Eluent: mobile phase leaving the column.

  • Retention time: the time for a particular analyte to pass through the system (from column inlet to detector) under set conditions.

Protein Purification: Based on Size and Shape

• Gel-filtration (size chromatography).

• Preparative gel electrophoresis (SDS-PAGE).

• Many proteins are multimers of more than one polypeptide.

• Multimers can be dissociated, leading to loss of structure/function.

• Proteins may have two “sizes”: native and denatured state.

• Gel-filtration normally deals only with native proteins.

• Electrophoretic procedures commonly involve separation of dissociated and denatured polypeptides.

Gel Filtration Chromatography

• Separates molecules based on their size and shape.

• Also known as size exclusion chromatography

Protein purification: based on net charge

• Ion-exchange chromatography

• Exploits overall charge of proteins

• Separation with highest resolution for native proteins

• Anion & cation exchangers

• immobilized charged groups that attract oppositely charged proteins

• net charge of a protein depends on pH

• positive at very low pH, negative at high pH

• zero at specific point in between = isoelectric point (pI)

• majority of proteins are negatively charged at neutral pH

Protein purification: based on surface features

• Surface features

• charge distribution and accessibility

• surface distribution of hydrophobic amino acid side chains

• net charge at given pH

• Hydrophobic chromatography

• surface distribution of hydrophobic residues determines solubility

• Selective precipitation

• solvent contains a low concentration of buffer salts

• change ionic strength, dielectric constant, pH, temperature & detergent content

Protein purification : based on bioproperties (affinity)

• particular biological property of protein is exploited

• Affinity chromatography

• Strong affinity between protein's binding site and its ligand

• immobilized ligand attracts protein from a mixture, while other molecules are washed away

• Fusion proteins (e.g. His-tag)

• strong affinity toward an immobilized ligand

• protein of interest don’t require any binding property of its own

• Immunoaffinity chromatography

• antibody directed against an epitope on protein surface is ligand that is used to pull out desired protein from mixture

• Immuno-affinity chromatography involves using an antibody specific to the protein of interest to capture and purify it.

Factors affecting efficiency of Column Chromatography

• Dimension of column:

  • Select suitable column dimension for application

  • Example: XK 50/30: 50 denotes inner diameter of column (mm) and 30 is length or height of column (cm)

  • Increased column length may improve separation

  • Optimization required for each process

• Particle size of the matrix:

  • small, medium and super fine range particles available

  • Decreasing particle size may improve separation

• Solvents:

  • should not affect stability of proteins or cause deleterious effects

  • should be compatible with column and matrix

  • Generally, a solvent with low viscosity is used – won’t affect the flow rate and thus separation

• Temperature:

  • proteins can degrade at high temperatures

  • Some columns can get damaged around 60°C

  • Large-scale protein purification processes mostly done at 4°C

  • Lab-scale protein purification usually at room temperature

  • Maintain lower temperatures for improved yield and efficiency

• Flow Rate:

  • Flow rate can be maintained using a peristaltic pump or AKTA systems (Protein purification controller)

  • Each chromatographic media or matrix tolerate specific maximum flow rates

  • Run column at Medium flow rate

  • very slow flow rate can lead to zone spreading

  • too fast flow rate can cause extensive tailing

• Packing:

  • Ensure proper packing of column

  • Degas matrix and buffers to remove air bubble trapped inside

  • Pour matrix/media into column in single step

  • Accurately calculate amount of matrix required for column

  • Place the column straight up

  • Check filters (mesh) and tubing connections before starting

  • Purge lines to remove air bubbles

  • Equilibration, washing, matrix selection, etc. also can influence the efficiency

  • process optimization required for improved results

  • Store chromatographic matrix in 20% ethanol

  • Wash with 3-7 column volumes of water to remove ethanol

Characterization of protein product

• Once a pure protein is obtained:

  • enzymatic analysis or test as a therapeutic agent

• Characterise protein in terms of structure and function:

  • Molecular weight (SDS-PAGE and/or gel filtration)

  • Spectral properties (UV spectrum, circular dichroism)

  • prosthetic groups (glycoproteins)

  • amino-terminal sequence analysis

• Functional proteins should have the appropriate function

• detailed kinetic characterization is required for most enzymes

Proteomics

• Proteome = entire protein complement in a given cell, tissue or organism

• Proteomics = large-scale study of proteins, particularly their structures, functions and interactions

• proteome differs from cell to cell and from time to time

• distinct genes are expressed in distinct cell types or in various stages in cell cycle

• In the past, mRNA analysis was used to define which proteins are being produced

• Don’t always correlate with protein content

• mRNA produced in abundance may be degraded rapidly

• post-translational modifications affects protein activities

• transcripts may give rise to more than one protein

• proteins may form complexes with other proteins or RNA molecules and only function in their presence

• Variation in protein degradation rate

Proteomics : protein analyses

• Two dimensional (2D) gels separate proteins first by iso- electric focussing and then by SDS-PAGE in 2nd dimension

• allows small differences in proteins to be visualized by separating a modified protein from its unmodified form

• Also identify differential production of proteins (in different tissue or in cells treated in a different way)

• Mass spectrometry at CAF

• quantitative and qualitative analysis of organic molecules

• GCMS analysis, LCMS analysis, accurate mass determinations, MALDI-TOF analysis and Proteomic analysis

• LTQ Orbitrap Velos - analyse 1000s of proteins in one run