Biochem Lecture 6

Size-based separations

- Dialysis - Gel filtration (Molecular Exclusion or Size Exclusion) - Ultracentrifugation - Centrifugal concentrators

Charged-based separations

- Ion Exchange chromatography

Electrophoresis

- SDS-PAGE - Isoelectric focusing - 2D Gel electrophoresis

Protein analysis by ultracentrifugation

1. The sedimentation velocity of a particle depends in part on its mass. A more massive particle sediments more rapidly than does a less massive particle of the same shape and density. 2. Shape, too, influences the sedimentation velocity because it affects the viscous drag. The frictional coefficient f of a compact particle is smaller than that of an extended particle of the same mass. Hence, elongated particles sediment more slowly than do spherical ones of the same mass. 3. A dense particle moves more rapidly than does a less dense one of the same mass because the opposing buoyant force (1 - ρ) is smaller for the denser particle. 4. The sedimentation velocity also depends on the density of the solution. (ρ). Particles sink when ρ < 1, float when ρ > 1, and do not move when ρ = 1.

Protein analysis by ultracentrifugation

1. The sedimentation velocity of a particle depends in part on its mass. A more massive particle sediments more rapidly than does a less massive particle of the same shape and density. 2. Shape, too, influences the sedimentation velocity because it affects the viscous drag. The frictional coefficient f of a compact particle is smaller than that of an extended particle of the same mass. Hence, elongated particles sediment more slowly than do spherical ones of the same mass. 3. A dense particle moves more rapidly than does a less dense one of the same mass because the opposing buoyant force (1 - ρ) is smaller for the denser particle. 4. The sedimentation velocity also depends on the density of the solution. (ρ). Particles sink when ρ < 1, float when ρ > 1, and do not move when ρ = 1. the image shows the range for proteins and all molecules in cells

Gel Filtration Chromatography

AKA size/molecular exclusion chromatography • Smaller proteins experience a higher mobile phase volume because they are able to enter pores of the stationary phase beads. • The elution volume is inversely related to the molecular weight • Chromatographic resolution depends on a number of factor including bead diameter, pore size, salt concentration, column volume, flow rate • Other factors constant, resolution increases with column volume • Protein must be freely soluble • Detergents necessary for membrane proteins • Typically 0.15 M salt necessary to prevent non-specific interactions between proteins and beads. • Volume of protein injected onto column must be <2% of column volume. column with agarose bead at the end and flowing buffer solution through column, loaded with 3 different size proteins large proteins are exludedf from bead pores, mediums can get in a little bit and small ones can get into the pores a lot protein solution must be 2% of the column volume so if a tube holds 100mL of column packing then you can load 2mL of solution if you have 1L of protein solution you need 50L of column to keep [rptins happy in a solution, use 15o milliM of salt (normal saline) which is usually used to keep non specific interactions from occurring and sometimes if the protein is sticky, it is necessary to use a detergent in the buffer

Ion Exchange chromatography

Charged-based separation A protein in a buffer at pH > pI will be negatively charged and able to bind an anion exchange resin. A protein in a buffer at pH < pI will be positively charged and able to bind a cation exchange resin Proteins may be eluted with a gradient of increasing salt (NaCl, or other) The elution order will depend on how tightly (charged) is a given protein Injection volume not limited image shows a - charged because with a mix of two proteins so the + charged proteins will stick to the beads -the - charged proteins will just flow through -even if you have a large volume protein, al; the ones that you desire will stick independent of the volume -you can elute the ones that are stuck by increasing the concentration of the salt

Ion Exchange chromatography

Charged-based separation • Ion exchange entails loading a protein mixture at a given pH in a low salt concentration buffer and washing the unbound material through the column • For cation exchange, positively charged proteins bind to the column, negatively charged proteins pass through • For anion exchange, negatively charged proteins bind the column, positively charged ones pass through • Bound proteins are eluted by a gradient of increasing salt concentration • Weakly bound proteins elute at a relatively low salt concentration, tightly bound ones at higher salt concentration • Proteins are focused by binding to the column. Thus, large volumes of solution may be applied to the column. notes: column packed with beads but this time beads are - charged and proteins are + charged - charged beads will stay bound to column and alluded + charged beads will undergo anionic exchange

Proteins must be extracted from their cell or tissue context for purification

Differential Centrifugation Treatment with Low or High Salt (osmotic lysis)

Myoglobin crystals and X-ray diffraction pattern

Electron Density Map used to build a 3-D structure so x-ray light can shine through

Circular Dichroism of Proteins

Figure 5. change in secondary structure in a detectable way caused by mutation

Omics

Genomics: organismal study of patterns of gene expression related to disease and developmental processes - Human genome project: all human genes sequenced - Approximately 30k human genes • Functional genomics: effort to make use of the vast wealth of data from the various genomics projects to understand gene and protein functions and interactions - Focuses on dynamic aspects of gene transcription, translation, and protein-protein interaction • Proteomics: large scale study of protein expression: functions and structures • Glycomics: Study of glycosylation of proteins and lipids on an organismal scale • Metabolomics: Study of the patterns of small molecule metabolites expressed in a biological system

How would mutation of a Leu to an Asp in hemoglobin affect its molecular weight? Its charge? Its pI?

MW would change but not very much Charge would change pI would change

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE)

Protein-SDS complexes migrate approximately according to mass • Mobility proportional to the logarithm of mass • This assumes that the ratio of bound SDS molecules per mass unit of protein is the same for all proteins • In reality, this ratio, and thus the relative mobility in the electric field, varies according to several factors • Hydrophobicity of polypeptide (membrane proteins migrate differently than soluble proteins) • Glycosylation: most proteins are glycosylated, and many glycans are acidic • Phosphorylation: ubiquitous signalling mechanism, lowers pI • Mature protein heterogeneity results in diffuse bands. notes: to determine protein molecular weight, one would plot a relative mobility, how far protein travels downs gel proteins being used are similar to the proteins used for MW calibrates if protein is highly decorated with sugar, it may not travel through gel in the same way plot scales shows an estimation of molecular weight

Gel Filtration Chromatography

Size Based AKA size/molecular exclusion chromatography or consists of agarose beads which are packed into a glass column which have pores protein molecules are passes through the column in solution the big molecules are too big to get into the pores and experience a small volume large molecules come out first for better separation, use a bigger column column requirement = protein must be soluble

Differential Centrifugation

Size Seperation Cells are disrupted in a homogenizer and the resulting mixture, called the homogenate, is centrifuged in a step-by- step fashion of increasing centrifugal force. The denser material will form a pellet (come out first) at lower centrifugal force than will the less-dense material. The isolated fractions can be used for further purification. you can see enzyme activity and & patterns of protein expression

Protein analysis by Ultracentrifugation

Sub-cellular fractionation (preparative) Analysis of protein mass, density, shape, binding (analytical) A particle will move through a liquid medium when subjected to a centrifugal force. A convenient means of quantifying the rate of movement is to calculate the sedimentation coefficient, s, of a particle where m is the mass of the particle, v is the partial specific volume (the reciprocal of the particle density), ρ is the density of the medium and f is the frictional coefficient (a measure of the shape of the particle). The (1 - ρ) term is the buoyant force exerted by liquid medium. Sedimentation coefficients are usually expressed in Svedberg units (S), equal to 10-13 s. The smaller the S value, the slower a molecule moves in a centrifugal field.

Amino acid analysis

Useful method for establishing that a given protein preparation has an amino acid composition that matches its theoretical sequence • Used in industry for recombinant protein-based products - Recombinant drugs - Antibodies - Animal feeds and additives • Requires a relatively large quantity of pure protein, primarily due to limitations of the hydrolysis step (high temperature, 6N HCl) ~300 pmol or 30 g of a 100 kDa protein notes: useful because we can calculate what the amino acid composition should be so when we measure it, it will tell us whether the protein is pure used by taking a protein sample, digested at low pH, all peptide bonds are hydrolyzed and converted to proteins then amino acid constants

Proteins are Amphiphilic Macro-Ions

Variable Properties •Solubility •Size •Charge •Hydrophobicity •Binding/adhesion if proteins are globular, remember that they have acidic and basic residues which are hydrophilic and tend to be located on the surface so there will patches of positively and negatively charge residues and even some amphiphilic regions this shows how proteins are amphiphilic macroions

X-ray crystallography

another way to determine protein structure a crystal is made of pure protein so an x ray light can be shine through it to determine the 3-D structure the challenge is to figure out how to make the protein solution turn into ordered crystals which can be subjected to the x ray light

Western Blotting

antibody based technique way to detect specific protein _ how much run an SDS phage gel lyse cell run them on SDS page transfer protein on membrane by gel electrophoresis put membrane i solution that contains antibody the protein will react with the antibody (particular and very specific) The antibody can be radio labeled or have a second antibody and be able to stain only the protein in question

ELISA: Enzyme-linked Immunosorbent Assay

antibody technique used to quantify targeted proteins, required high quality antibodies

Cells may be disrupted by treatment with low or high salt (osmotic lysis)

cells may be disrupted by treatment with low or high salt (osmotic lysis) and the protein in question purified from the cytosol or membrane fraction.

Proteomics: using genomic sequences to identify proteins

conceptually take all of a cell lysate, digest proteins & analyze know DNA sequence to predict the mass of the proteins in solution and use info to interpret the proteins masses

The cytoplasm

cytoplasm is dense proteins interact with one another proteins stay soluble as a result of numerous homeostasis interactions

What does relative mass measurement (a singel electrophoresis) tell the biochemist?

density = quantity of amino acid purity = one band mw = location

NMR is a form of spectroscopy using magnetic fields and radio frequency photons

detects protons based on spin

What are the advantages to Edman degradation?

direct sequence of amino acid starting from N-terminus

NMR Spectroscopy informs about organic structure

each proton will have own characteristic pattern can determine overall fold reminds us that proteins are moving and flexible

Ordering of peptide fragments by overlapping sequences produced by specific proteolysis of a peptide

example of how proteins can be sequenced using chemical methods (overlapping sequence)

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE)

gel made of acrylamide to use one runs a standard curve of proteins of know MWs density of gel depends on density of linking chains density of band indicates how much protein may be present

Reversed phase chromatography

great way to operate proteolytic or tryptic peptides • Reversed phase HPLC is a ubiquitously useful means of separating proteolytic peptides using a gradient from low to high percent organic • Peptides bind to C18 (very hydrophobic tail) stationary phase in low organic solvent, elute with a gradient of increasing organic content • Trifluoroacetic acid (TFA) is an ion-pairing agent that prevents charge-based interactions between peptides and stationary phase • Proteins are denatured by TFA and organic • Useful as a means for separating intact proteins for structural studies. notes: 1. one equilibrates the column in water 2. binds peptides to column 3. peptides stick 4. increasing gradient of acetonitrile (from water) 5. as % of acetonitrile increases, peptides elute in order according to hydrophobicity more hydrophobic and large elutes last

Proteomics: discovery of proteins (qualitative)

how to identify thousands of proteins in an hour protein solution can be a cplete cell lysate 7 digested with trypsin to get a mixture of many peptides the many peptides are speerates using reverse phase HPLC in which most hydrophilic peptides come out first there can be numerous peptides for single point which will be detected by using mass sect each individual peptide will reveal its sequence using tandem MS

Examples of ion-exchange chromatography

image shows elution by changing pH F has lowest electropoint C has highest electropoint the proteins to elute is the one that is least cationic and the most cationic will ne the last to elute (we have to raise the pH highest to get last protein to have - charge character if you want to separate proteins like hemoglobin, we can use ion exchange chromatography

Coordinated differentiation and morphogenesis in the mammalian tooth

image shows pattern of different paracrine functions as a function of the development from the rat teeth over 14 days we are interested in the development of teeth and recapitulation of teeth towards treating dental problems when there is a problem with the bone surrounding the teeth, it may not be possible reformation of teeth may not be possible until the bone is stimulated to grow an understanding of the paracrine factors (proteins) will allow us to manipulate cell growth in tissue

Dialysis

involves taking protein solution and putting it inside a membrane with a controlled pore size which is in a buffered solution as it dialyzes, small molecules go through and the big ones don't so the concentration of the small molecules is the same inside and outside molecules must differ greatly in size • Protein molecules (red) are retained within the dialysis bag, whereas small molecules (blue) diffuse into the surrounding medium. • Separation of large from small molecules • Change of the solution properties (desalting, crystallization) • Solubility, recovery serious issues

Why is amino acid analyst used for proteins?

its a good way of proving that you have the right protein purity if the amino acid is pure then it should be the same as the one you calculate SDS will tell you how pure the protein is

What does accurate mass measurement( as in mass spectrometry) of a protein mass tell the biochemist?

mass sept tells you ecxcatly that mass of the protein shifted mass will show mutation accurate mass will show wild type proteins

Size based separations: Gel-filtration chromatography Graph

measures time and absorbance of solution coming out of column not all proteins are completely resolved 2,34, are not all completely separate all peptide bonds absorb at 220 nm light the only side chains that absorb light at 280 are tyrosine and tryptophan

Circular Dichroism: fixed wavelength measurement for determination of protein melting point (protein stability)

melting curves used to see if protein is in a native state

Affinity chromatography

method of separating biochemical mixtures based on a highly specific interaction between antigen and antibody, enzyme and substrate, or receptor and ligand. used for purifying biological molecules within a mixture by exploiting molecular properties Biological macromolecules such as enzymes and other proteins, interact with other molecules with high specificity through several different types of bonds and interaction (hydrogen bonding, ionic interaction, disulfide bridges, hydrophobic interaction) The high selectivity of affinity chromatography is caused by allowing the desired molecule to interact with the stationary phase and be bound within the column in order to be separated from the undesired material which will not interact and elute first The molecules no longer needed are first washed away with a buffer while the desired proteins are let go in the presence of the eluting solvent (of higher salt concentration). This process creates a competitive interaction between the desired protein and the immobilized stationary molecules, which eventually lets the now highly purified proteins be released saves a lot of steps

What Does Circular Dichroism Tell Us About Structure?

molar absorptivity of left or right handedness of polarized light used to detect changes in structure caused by mutation or perturbed structure (detection method for protein structure)

Circular dichroism and protein structrure

optical absorbance technique UV absorbance using a special type of polarize light

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE)

protein purity, homogeneity, distribution

Use of Amino Acid Analysis in Diagnosis of Disease

purified proteins can show problems associated with levels of amino acids in plasma or urine in disorders

The Edman reaction for sequencing peptides

reacts with N-terminal amino acid to make a phenol this-cyan derivative which will break off the N-term amino acid to form phenylthiohydantoin one identifies which amino side chain it is using the retention time of a chromatography step and these steps repeat for the next amino acid one cycle per reaction

What resources are needed to successfully use omics technologies?

requires infrastructure & needs to be a balance of comic techniques

Protein analysis by Ultracentrifugation

size based separation use to purify proteins or determine MW to purify: create a density gradient in tube using a cell with high percent sucrose on one side and low percent on other so you can make a gradient then layer sample at the top, spin it at high velocity. proteins will move appropriate density of solution most dense will move the most then you can fractionate the proteins protein mass can be measured accurately by sedimentation equilibrium mass measurement under native conditions, applicable to multi protein complexes

What technique would be used to separate a 500kDa protein from a 50 kDa protein

size exclusion ultracentrifugation

Two-dimensional gel electrophoresis The Principle of Isoelectric Focusing

sized based separation A pH gradient is established in a gel before loading the sample. (A) The sample is loaded and voltage is applied. The proteins will migrate to their isoelectric pH, the location at which they have no net charge. (B) The proteins form bands that can be excised and used for further experimentation. IEF • Isoelectric point = pI = pH where z = 0 • Loading an issue notes: drizzle protein solution with a pipet and turn on voltage proteins will migrate according to charge proteins will travel to where they have zero charge or where pH = pI

Specificity of some polypeptide cleaving reagents

specificity for polypeptide cleavage reagent chopping protein up into peptides and sequencing the peptides (shot gun experiment) you have a number of different reagents available for chopping up protein

Liquid chromatography-mass spectrometry

sprayed solution through novel apply electric field spray peptides into mass spec mass pec determines mass of peptides as it elutes

How might comics technologies influence dental practice?

studying diseases why can't teeth be regenerated many problems associate with age better understanding and ways to shed light on these problems

Typical Proteins Recovery

table shows % recovery of different protein's MWW for a molecule to pass through

INDRECT ELISA

take antigen (molecule you are trying to deter) & add antibody which is specific to the antigen for it to bind then it detects how much antigen is present by adding a second antibody which is specific to the constant region of 1st antibody antibody has an enzyme on it which creates a color change concern is what if antibody isn't specific because its an indirect detection

What is dialysis used for

to get rid of salts or other small molecules from our protein solution

Wha tis the difference between traditional biochemistry and the emerging systems-wide "omics" approaches (genomics, proteomics, glycomics, metabolomics) ?

traditional biochem: isolate tissue, extract proteins, purify proteins emerging systems-wide identify proteins in tissue at once

SANDWICH ELISA

two monoclonal antibodies to the same antigen or protein 1st monoclonal antibody is bound to the well antigen is applied and is not immobilized second antibody is also specific for antigen and has enzyme that causes the color change now detecting the direct binding between the antibody and antigen overcomes problems associated with indirect natured substrate of enzyme is added color change can be used to quantify the protein

High performance liquid chromatography

used for separating peptides and purifying • Chromatographic resolution increases as the size of the chromatographic beads decreases • The pressure required to push solvent (mobile phase) through the packed beads increases as their size decreases • High performance liquid chromatography (HPLC): a pumping system that pushes solvent through a column packed with small beads (approximately 5 microns). • All tubing, columns, made of stainless steel to withstand pressures up to 2500 psi or so. • Higher performance and cost than conventional low pressure liquid chromatography. • Typically one begins with low pressure chromatography to concentrate the protein and then switches to HPLC for later steps High performance : small beads made out of silica glass so they can maintain structure at high pressure small beads are better able to separate peptides

Centrifugal concentrators:

works well for removing salts or small molecules from proteins has a dialysis membrane at the bottom solution is put on the top and solution is forced through the membrane using centrifugation so we can dialyze faster note that a dialysis membrane is the same before and after. The volume of the solution hasn't changed but in the centrifuge cell, we are changing the volume and increasing the protein concentration as a result of the effects of centrifugation • Centrifugal force pushes solution through a membrane with size selective pores • Useful for concentrating, desalting proteins • Also dialysis concentrator cells

Informational value of amino acid sequences

• BLAST (Basic Local Alignment Search Tool) www.ncbi.nih.gov computes alignments for a given sequence to known sequences • Does a protein belong to a given family? • Evolutionary trees based on protein sequence similarity • Are there internal repeats in a protein sequence? Evidence for duplication of primordial genes • Sequence data may be used to generate antibodies against a protein of interest (Western blotting, immunohistochemistry, immunoprecipitation, ELISA) • AA sequences useful for design of DNA probes specific to the corresponding gene. • What are the post-translational modifications on the protein/peptide?

Amino acid analysis

•Is the protein pure? •Is it the right protein? •Does it have the expected AA composition?

NMR Spectroscopy

•Nuclear magnetic resonance measures the environment of protons and other nuclei (13C, 15N, 31P) •CH, NH, OH, COOH, etc •NMR experiments determine distance constraints between NMR active nuclei in biomolecules •High concentration, high purity protein needed •Able to measure protein dynamics •Limited ability to solve structures of very large proteins •Expression of isotope enriched proteins to maximize NMR sensitivity •Requires a very large magnet (900 MHz NMR spectrometer, above) •Resource and computationally intensive >6000 protein structures solved