2. 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 AA
common to all amino acids*
*except proline, in which the R group forms a
ring structure by binding to the amino group
3. Amino acids can act as Acids and Bases
• When an amino acid is dissolved in water, it exists in solution as the dipolar
ion, or zwitterion (German for “hybrid ion”).
4. 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.
5. The Henderson-Hasselbalch Equation
• In chemistry, the Henderson–Hasselbalch equation describes the derivation of pH as a
measure of acidity (using pKa, the acid dissociation constant) in biological and chemical
systems.
• The equation is also useful for estimating the pH of a buffer solution and finding
the equilibrium pH in acid-base reactions (it is widely used to calculate the isoelectric
point of proteins).
Here, pKa is − log(Ka) where Ka is the acid dissociation constant
Dissaciation 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-).
6. Calculation of pI using Titration curves
•Titration curves are produced by monitoring the pH of given volume of a sample
solution after successive addition of acid or alkali
•The curves are usually plots of pH against the volume of titrant added or more
correctly against the number of equivalents added per mole of the sample
7.
8.
9. 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.
10. Principle of Two dimensional gel electrophoresis
First Dimension:
Denaturing isoelectric
Second Dimension:
focusing in presence SDS polyacrylamide
of 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
11. 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.
12. 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
13. For hydrophobic proteins
Membrane proteins do not easily go into solution. A lot of optimization work is
required.
1. Thiourea procedure
2. SDS procedure
3. New zwitterionic detergent and sulfobetains
14. Problems with 2D-SDS-PAGE
1. 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.
15. 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.
17. General detection methods
Organic dye- and silver-based
methods
Coomassie blue
Silver
Radiactive labeling methods
Reverse stain methods
Flourescence methods
18. In-Gel Digestion
Transfer the gel slice to microtube
Spot picker picks the
protein of interest
from 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.
19. There are two techniques in predominant use
(1) Fingerprinting by MALDI-TOF and
(2) de novo sequencing by ESI/MS/MS
Fingerprinting works on the following principle. Unique proteins have unique peptide sequences
Thus 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 Activity
molecular weights of each of these fragments are detected in the MALDI-TOF and accurately
describe the fingerprint, this is possible because MALDI -TOF is a gentle ionization method. One
can predict the peptide sequence of the corresponding peptide from genomic data as well as the
resulting tryptic digest fragments and fragment masses. Thus a search of the experimentally
determined values against databases can provide the identity of the protein in question. This is
demonstrated in the figure below.
<< Previous Page | Next Page>>
1 | 2 | 3 | 4 | 5 | 6 | 7
Protein 1 fingerprint Protein 2 fingerprint
20. Different strategies for proteome purification and
protein 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.
38. 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 ?
39. Plasma is the largest and deepest version of the human
proteome
• Largest = Most proteins
• Deepest = Widest dynamic range
40. The major plasma proteins. This image demonstrate the high dynamic range of proteins
present in a plasma sample.
41.
42.
43.
44. Plasma Proteome Database
• Base list of ~450 proteins reported in “non-proteomics” literature as measured/detected in
plasma 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-based
prediction
– Secreted
– Extracellular domains of plasma membrane proteins
• Forms a basis for application of multivariate (pattern) discovery
focused on “known” proteins
45. Figure . 2DGE protein profiles for crude (70 µg protein) and depleted (100 µg protein) serum samples. Agilent’s Multiple
Affinity 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.
48. 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 .
49.
50. Immunity Affinity Subtraction Chromatography (IASC)
Anion Exchange Chromatography (AEC)
Size Exclusion Chromatography (SEC)
2D gel electrophoresis
Mass Spectrometry
51. The Current Phase of Plasma Proteomics Employs
Multi-Dimensional (>2D) Approaches (e.g., 3-D
Chromatography + 2-DE + LC/MS)
52. Retinal Binding
Toponin protein
Human serum protein pattern after removal of several abundant proteins by IASC
as visualized in a CBB-stained 2-DE gel.
53. Fig a Fig b
2-DE spot positions of MS-identified proteins in serum following 3-DLC fractionation. The CBB-stained gel N183F
corresponds to a fraction eluted from the POROS HQ column (AEC), which – upon fractionation by SEC – eluted in the Mr
range of 95-110kDa Figa, 75–85 kDa Figb.
54. Categories of proteins identified in human serum. The sizes of the pie segments (with adjacent numbers) are proportional to
the number of nonredundant protein annotations for the following serum protein categories. 1. Classical plasma proteins in
circulation; 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 – exported
into extracellular fluids; 4. Cell surface membrane proteins; 5. Intracellular proteins, presumably leaking from cells and
tissues into blood plasma; 6. Uncategorized (proteins for which cellular designations are unknown).
56. 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.
59. 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
60. 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
61. 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 an
individual
– Reduce cost of disease management through early intervention to
prevent progression
• Comprehensive health monitoring: disease states using one
sample type (serum/plasma)
– Proteomics increases pool of potential marker proteins
– Multiprotein markers (panels) provide greatly increased statistical
power (more accurate diagnosis)
64. • 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)
65. As IEF is based on non-denaturing conditions, it is well suited for purification of proteins, which
will be obtained in active form and natural shape. An important application is pre fractionation of
proteins before loading onto pI-strips.
Pre fractionation is especially useful to circumvent problems of different abundance. Low
abundance proteins may be separated from others and pre concentrated, so that they can be
resolved and visualized in 2D-electrophoresis.
Thus the combination of pre fractionation step with 2D-electrophoresis may heavily extend the
number of proteins which can be separated, detected and characterized.
Preparative IEF has been performed with large and thick gels, but through the last few years
several 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 is
partitioned by membranes. For liquid phase electro focusing typically free carrier ampholytes are
used.
66. 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.
67. 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 desired
Cathode 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
69. 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.
70. • 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
71. Advantages over other methods:
•Apart from the excellent resolving power, FFE has a second important advantage over
methods like 2D gel electrophoresis in that samples can be injected, separated and
collected continuously.
•There is unlimited loadability, so that significant amounts of each fraction can be
amassed. In the case of intact proteins, as the authors point out, this is an ideal scenario
for pre fractionating before top-down sequencing, where whole proteins are analyzed by
mass spectrometry.
•In addition, the peak capacity was calculated to be about 6720, compared with 100-130
for a single RP-HPLC analysis of proteins, or 2640 from a recent report combining
capillary isoelectric focusing with RP-HPLC.
•Apart from intact proteins, the combined FFE-RP-HPLC system is equally suitable for
the analysis of small molecules. It is envisaged that it will play a major role in the
search for new biomarkers of disease, emphasising its flexibility.
72.
73.
74.
75. Applicability
Conventional machines may be operated with a low ionic strength uniform electrolyte
in the zone electrophoretic mode only.
Peptides, proteins, DNA, viruses, organelles, bacteria or cells can be separated at
resolutions of 3-5% of their electrophoretic mobilities and a throughput of up to 50 mg
protein or 20 million cells per hour may be achieved.
Highly developed modern machines may be operated continuously or at intervals with
segmented electrolyte in various modes and with buffers containing up to 60 mM ions.
