Isoelectric Point (pI) The isoelectric point (pI), sometimes abbreviated to IEP, is the pH at which a particular molecule or surface carries no net electrical charge. General Structure of an Amino acid different for each AAcommon to all amino acids* *except proline, in which the R group forms a ring structure by binding to the amino group
Amino acids can act as Acids and Bases• When an amino acid is dissolved in water, it exists in solution as the dipolarion, or zwitterion (German for “hybrid ion”).
A zwitterion can act as either an acid (proton donor):or a base (proton acceptor):Substances having this dual nature are amphoteric and are often called ampholytes.
The Henderson-Hasselbalch Equation• In chemistry, the Henderson–Hasselbalch equation describes the derivation of pH as ameasure of acidity (using pKa, the acid dissociation constant) in biological and chemicalsystems.• The equation is also useful for estimating the pH of a buffer solution and findingthe equilibrium pH in acid-base reactions (it is widely used to calculate the isoelectricpoint of proteins). Here, pKa is − log(Ka) where Ka is the acid dissociation constantDissaciation constant, Ka = [A-][H+]/[HA]pH = -log[H+] When [A-] = [HA]:pKa = -log(Ka) pH = pKa + log(1) pH = pKa + 0 pH = pKa pka is the pH at which a functional group exists 50% in its protonated form (HA) and 50% in its deprotonated form (A-).
Calculation of pI using Titration curves•Titration curves are produced by monitoring the pH of given volume of a samplesolution after successive addition of acid or alkali•The curves are usually plots of pH against the volume of titrant added or morecorrectly against the number of equivalents added per mole of the sample
Calculation of pI for a peptide Neutral side chain aminoacid: Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionione, Serine, Threonine Charged side chain aminoacid: Aspartate, Glutamate, Cysteine, Tyrosine, Histidine, Lysine, Arginine If a polypeptide chain composed only of neutral side chains aminoacids, then pKa of only N-terminal and C-terminal of the ploypeptide is neededing to be considered for pI determination.
Principle of Two dimensional gel electrophoresisFirst Dimension: Denaturing isoelectric Second Dimension: focusing in presence SDS polyacrylamideof urea, Nonidet NP-40 gel rod equilibrated gel electrophoresis in vertical gel rod in SDS equilibration buffer sample pH 3 pH 10 pH 10 pH 3 Separation according to Separation according to Isoelectric Point (charge) Molecular Weight (mass) Principle according to P.H. O’Farrell (1975)1) High molar (8 mol/L) urea, 2) Non-ionic or zwitterionic detergent• single conformation of a protein for protein solubility• For protein solubility 3) Carrier ampholyte mixture• prevents protein aggregates and For pH gradient hydrophobic interactions 4) DTT prevents different oxidation steps
Immobilized pH gradient (IPG) Acrylamido buffers: Immobiline CH2=CH-CO-NH-R, R contains a carboxylic or a tertiary amino group. The buffering Groups are covalently coupled to the acrylamide molecules.High reproducibilty compared to tube gel.No cathodic drift.Sample volumes are high.
Immobilized pH gradient (IPG)• Few problems of carrier ampholytes were solved in immobilized pH gradients (IPG).• IPGs are generated by buffering acrylamide derivatives,which are copolynerised with the gel matrix.• The acrylamide derivatives containing the buffering groups are called immobiline. CH2 CH C O + R (ampholytes) NH2 R= 3 weak acids (carboxyl groups) with pKs 3.6, 4.4 & 4.6 4 bases (tertiary amino groups) with pKs 6.2, 7.0, 8.5 & 9.3
For hydrophobic proteinsMembrane proteins do not easily go into solution. A lot of optimization work is required.1. Thiourea procedure2. SDS procedure3. New zwitterionic detergent and sulfobetains
Problems with 2D-SDS-PAGE1. Difficulty of performing completely reproducible analyses - Differences in protein migration in either dimension could be mistaken.2. Relative incompatibility of some proteins with the IEF step - Hydrophobic proteins: precipitation & aggregation “smearing” - Post-translational modification of proteins: pI change spot “trains”3. Relatively small dynamic range of protein detection - Less abundant proteins frequently cannot be detected. - ~3000 proteins out of ~4000 expressed genes were not detected in yeast.
Strips available in dry forms: have to be rehydrated before use.Sample can also be rehydrated alongside.Cup loading used after passive rehydration in the tray.
General detection methods Organic dye- and silver-based methods Coomassie blue Silver Radiactive labeling methods Reverse stain methods Flourescence methods
In-Gel Digestion Transfer the gel slice to microtubeSpot picker picks theprotein of interestfrom the gel Destain gel slice with Acetonitrile and Ammonium bicarbonate solution.2-D Gel Remove destaining solution and dry the gel in Speed Vaccum. Rehydrate with a protease solution. Incubate for 6 hrs to overnight at 37°C.
There are two techniques in predominant use(1) Fingerprinting by MALDI-TOF and(2) de novo sequencing by ESI/MS/MSFingerprinting works on the following principle. Unique proteins have unique peptide sequencesThus if two different proteins are treated with trypsin, which cleaves at specific basic peptides,each protein will give a unique set of peptide fragments - hence the term "fingerprint". The Protease Activitymolecular weights of each of these fragments are detected in the MALDI-TOF and accuratelydescribe the fingerprint, this is possible because MALDI -TOF is a gentle ionization method. Onecan predict the peptide sequence of the corresponding peptide from genomic data as well as theresulting tryptic digest fragments and fragment masses. Thus a search of the experimentallydetermined values against databases can provide the identity of the protein in question. This isdemonstrated in the figure below.<< Previous Page | Next Page>> 1 | 2 | 3 | 4 | 5 | 6 | 7 Protein 1 fingerprint Protein 2 fingerprint
Different strategies for proteome purification andprotein separation for identification by MS A. Separation of individual proteins by 2-DE. B. Separation of protein complexes by non-denaturing 2-DE (BN-PAGE) C. Purification of protein complexes by immuno- affinity chromatography and SDS-PAGE. D. Multidimensional chromatography. E. Organic solvent fractionation for separation of complex protein mixtures of hydrobhobic membrane proteins.
Two D gel electrophoresis : a question of choice ?1. Isoforms and post-translational 1. Basic proteins are not well modifications displayed. represented.2. High resolution, particularly after pre- 2. Hydrophobic and membrane proteins fractionation. not seen.3. High throughput, parallel runs. 3. Recovery of proteins from the gels is not efficient.4. Crude samples tolerance. 4. Gel to gel variation .5. Multiple detection, blotting, applicable. 5. No equivalent of PCR in proteomics.6. Efficient fraction collector. 6. Efficient protein detection ?
Plasma is the largest and deepest version of the human proteome • Largest = Most proteins • Deepest = Widest dynamic range
The major plasma proteins. This image demonstrate the high dynamic range of proteins present in a plasma sample.
Plasma Proteome Database• Base list of ~450 proteins reported in “non-proteomics” literature as measured/detected inplasma or serum• Additional experimental data being added– Three sets of 300-600 proteins each from proteomics surveys (2-D gels + MS/MS; LC/LC-MS/MS)• A non-redundant list has been derived and the accessions are being classified by function/source• Initial results: 1,158 distinct proteins (excluding Ig’s)• Proteins potentially detectable in plasma will be added through in silico genome-basedprediction– Secreted– Extracellular domains of plasma membrane proteins• Forms a basis for application of multivariate (pattern) discoveryfocused on “known” proteins
Figure . 2DGE protein profiles for crude (70 µg protein) and depleted (100 µg protein) serum samples. Agilent’s MultipleAffinity Removal Column was used to deplete six high-abundance proteins (albumin, IgG, IgA, α1- antitrypsin, transferrin,and haptoglobin), corresponding to a removal of 85% of total protein content. Image courtesy of AstraZeneca.
Multidimensional Protein Identification Technique (MudPIT)• Disadvantages associated with two-dimensional gel electrophoresis can be alleviated in MudPIT.• MudPIT uses two chromatography steps interfaced back to back in a fused silica capillary.• The advantage of this is that the band broadening associated with many chromatographic steps is avoided and also the capillary can be placed directly into the ion source of a mass spectrometer maximizing sensitivity.• Chromatography proceeds in steps with increases in salt concentration used to free peptides from the cation-exchange resin after which they bind to a reversed phase resin.• A typical reversed phase gradient to increasing hydrophobicity is then applied to progressively elute peptides from the reversed phase packing into the mass spectrometer.• Typically this mass spectrometer will be an tandem electrospray, so peptides are ionized in the liquid phase, separated in a primary mass spectrometer, broken up using collision induced dissociation and analyzed again .
Immunity Affinity Subtraction Chromatography (IASC) Anion Exchange Chromatography (AEC) Size Exclusion Chromatography (SEC) 2D gel electrophoresis Mass Spectrometry
The Current Phase of Plasma Proteomics Employs Multi-Dimensional (>2D) Approaches (e.g., 3-D Chromatography + 2-DE + LC/MS)
Retinal Binding Toponin proteinHuman serum protein pattern after removal of several abundant proteins by IASCas visualized in a CBB-stained 2-DE gel.
Fig a Fig b2-DE spot positions of MS-identified proteins in serum following 3-DLC fractionation. The CBB-stained gel N183Fcorresponds to a fraction eluted from the POROS HQ column (AEC), which – upon fractionation by SEC – eluted in the Mrrange of 95-110kDa Figa, 75–85 kDa Figb.
Categories of proteins identified in human serum. The sizes of the pie segments (with adjacent numbers) are proportional tothe number of nonredundant protein annotations for the following serum protein categories. 1. Classical plasma proteins incirculation; 2.Proteins in the extracellular matrix or secreted into body fluids other than plasma; 3. Vesicular proteins(including endoplasmic reticulum, lysosomes, peroxisomes, Golgi apparatus) also – presumably or knowingly – exportedinto extracellular fluids; 4. Cell surface membrane proteins; 5. Intracellular proteins, presumably leaking from cells andtissues into blood plasma; 6. Uncategorized (proteins for which cellular designations are unknown).
The Plasma Proteome as Diagnostic Tool•Contains Specific import and export products of nearly all cells in the body.•Contains debris from dead and dying cells ( non-apoptotic death).•Large dilution volume: ( Approx.2.5mL and 12 L of extracellular fluid).•Contains a high proportion of very heterogeneous glycoproteins.•Temporally dynamic:•Collected routinely for diagnosis.
Challenges Facing Marker/Diagnostic Proteomics• Translation into diagnostic tests– Lack of a protein measurement platform geared to validation (high-throughput, low-cost)– Access to large, well-organized sample sets for validation– Falling rate of new protein tests over last decade– Low expectation of diagnostic profitability impairs commercial investment
Factors Supporting Rapid Discovery of Improved Markers• Analytical advances in resolving proteins in plasma• Assembly of a database of candidate marker proteins• Development of protein panels instead of single protein tests.• Understanding of genetics to improve marker interpretation
Opportunities in Diagnostic Proteomics• Monitoring drug and drug vs disease effects– Clinical trials and routine patient monitoring– Surrogate markers, disease classification, response verification• Early detection (and intervention)– Exploit higher test sensitivity differences over time within anindividual– Reduce cost of disease management through early intervention toprevent progression• Comprehensive health monitoring: disease states using onesample type (serum/plasma)– Proteomics increases pool of potential marker proteins– Multiprotein markers (panels) provide greatly increased statisticalpower (more accurate diagnosis)
• Performed mainly in solution: liquid-phase IEF• pH gradient is generated with soluble ampholytes (polycarboxylic acid compounds)• Advantages of liquid-phase IEF - Large sample capacity - Easy handling (compared to gels)
As IEF is based on non-denaturing conditions, it is well suited for purification of proteins, whichwill be obtained in active form and natural shape. An important application is pre fractionation ofproteins before loading onto pI-strips.Pre fractionation is especially useful to circumvent problems of different abundance. Lowabundance proteins may be separated from others and pre concentrated, so that they can beresolved and visualized in 2D-electrophoresis.Thus the combination of pre fractionation step with 2D-electrophoresis may heavily extend thenumber of proteins which can be separated, detected and characterized.Preparative IEF has been performed with large and thick gels, but through the last few yearsseveral new formats of instruments for preparative focusing have been developed. For those,preparation is based on liquid phase electro focusing within a chamber that in some cases ispartitioned by membranes. For liquid phase electro focusing typically free carrier ampholytes areused.
Preparative IEF (Rotofor.)• polyester screens separate chamber into 20 compartments• fractions rapidly harvested following electrofocusing The Rotofor cell is a preparative isoelectric focusing (IEF) apparatus, in which IEF is performed entirely in free solution. Electrofocusing in Rotofor cell has been described as well-suited for use at any stage of a purification scheme. However, it has some important limitations in resolving complex mixtures of proteins. This paper describes the advantages and disadvantages of using the Rotofor cell in purification protocols. - The focusing cell is divided by permeable membranes Into a series of chambers. - After focusing step, the chambers are emptied by a vacuum sipper into separate tubes. - The entire protein mixture is separated into 12~20 fractions.
Rotofor Solutions Rotofor Tissue Extraction Medium (60 ml for large Rotofor chamber) 10% glycerol 6 ml pH 3-10 ampholytes 1.5 ml 10 mM MOPS (pH 7.2) 52.5 ml add neutral detergent if desiredCathode Buffer - 0.1 M H3PO4 - 2.3 ml phosphoric acid plus 198 ml ddH2O Anode Buffer - 0.1 M NaOH - 0.8 g plus 200 ml ddH2O
Free Flow Electrophoresis (FFE)• Free Flow Electrophoresis (FFE) is an electrophoresis procedure working continuously in the absence of a stationary phase (or solid support material such as a gel).• It separates preparatively charged particles ranging in size from molecular to cellular dimensions according to their electrophoretic mobilities (EPMs) or isoelectric points (pIs).• Samples are injected continuously into a thin buffer film flowing through a chamber formed by two narrowly spaced glass plates.• Perpendicularly to the electrolyte and sample flow, current may be applied while the fluid is flowing (continuous FFE) or while the fluid flow is transiently stopped (interval FFE).• The sample and the electrolyte used for a separation enter the separation chamber at one end and the electrolyte containing different sample components as separated bands is fractionated at the other side.• At the end of the chamber, the fractionated flow is collected in a series of 96 capillaries and deposited in a 96-well plate.• The well plates can be analyzed by gel electrophoresis or mass spectrometry but in the new technique, they are analyzed by RP-HPLC fitted with UV and fluorescence detectors for maximizing the detection capability.
• The data from FFE and HPLC were represented in a 2D format, plotting the pI (from FFE) against the hydrophobicity (from the HPLC retention time).• The resolution attained by FFE is high, at 0.02 pH units, and there are no physical barriers for the analytes, so low-molecular mass peptides or large proteins are equally amenable.• Once the compounds have been fractionated in the second step, by HPLC, selected compounds can be taken for further analysis, for example by mass spectrometry
Advantages over other methods:•Apart from the excellent resolving power, FFE has a second important advantage overmethods like 2D gel electrophoresis in that samples can be injected, separated andcollected continuously.•There is unlimited loadability, so that significant amounts of each fraction can beamassed. In the case of intact proteins, as the authors point out, this is an ideal scenariofor pre fractionating before top-down sequencing, where whole proteins are analyzed bymass spectrometry.•In addition, the peak capacity was calculated to be about 6720, compared with 100-130for a single RP-HPLC analysis of proteins, or 2640 from a recent report combiningcapillary isoelectric focusing with RP-HPLC.•Apart from intact proteins, the combined FFE-RP-HPLC system is equally suitable forthe analysis of small molecules. It is envisaged that it will play a major role in thesearch for new biomarkers of disease, emphasising its flexibility.
ApplicabilityConventional machines may be operated with a low ionic strength uniform electrolytein the zone electrophoretic mode only.Peptides, proteins, DNA, viruses, organelles, bacteria or cells can be separated atresolutions of 3-5% of their electrophoretic mobilities and a throughput of up to 50 mgprotein or 20 million cells per hour may be achieved.Highly developed modern machines may be operated continuously or at intervals withsegmented electrolyte in various modes and with buffers containing up to 60 mM ions.