Definitions
Constitutive proteome :All known proteins found in a normal cell
Differentially expressed proteins: Proteins produced in response to disease, drug treatment, toxic agents, growth
Functional genomics: Connecting gene to function
Proteome :Total set of proteins expressed in a given cell at a given time (i.e., dynamic state)
Proteomics: Proteomics is the large-scale study of proteins, particularly their structures and functions.
Why Proteomics
-Correlation between mRNA and expressed protein level only 0.48.
-Proteome is dynamic
–under different conditions, eg. cell cycle, stress, pathological
–PTM and alternative splicing
2DE
Principles
-1st dimesnsion: IEF: separation based on pIs in a pH gradient frm anode to cathode. (Equilibrium method)
-2nd dimension: SDS electrophoresis: separation according to MW. (Dynamic method)
Some times Liquid chromatography is performed 1st to produce fractions which undergoes 2DE.
Establishing pH gradient
-Ampholytes pH gradients
–Batch to batch reproducibility of the carrier ampholytes is inadequate
–Carrier ampholytes gradients are unstable
–Limited protein loading capacity
–Gradient drift causes lack of reproducibility
–Gradient drift causes loss of basic and acidic proteins
–Soft gel rods have poor size stability
–Personal skill influences results
-Immobiline pH gradients (dry strips)
–High reproducibility
–No gradient drift, true equilibrium method
–High loading capacity
–Possibility of sample application by rehydration
–Ready-made strips
–Easy handling
Mass spectrometery
Advantages
-Fast
-Sensitive
-Detect PTM
-Accurate estimate of molecular masses (PMF)
Principles
-intro sample into MS
-ionisation
-analyze mass/charge of ions
-determine of MW
Biological MS
Heating will decompose biological molecules, so MALDI and ESI-MS are used.
Matrix-Asssired LAser Desorption/Ionization
-Proteins placed in a light absorbing matrix are irradiated by a laser pulse that desorb and ionize protein and matrix into the vaccum system
Electrospray Ionization MS
-Protein solutions are forced directly from the liquid to the gas phase by passing through a charged needle, producing ions protonated at basic amino acids (lysine and arginine)
MS sequencing
Hooking two MS in tandem
-1st MS separates protein digest (usually trypsin as C terminus generated will yield y-ion series)(based on m/z differences) and select one peptide for sequencing
-On the way the peptide is fragmented by collision with Helium/Argon gas (collision induced dissociation, CID)
-Fragments analyzed by the 2nd MS
-In practice, not all b and y fragment ions are present in detectable levels, and internal fragment ions are also present
Bottom-up MS
-High sensitivity, mass accuracy, resolution and dynamic range
-High expense and operational complexity limit routine use in proteomics research.
Usage of different solubilities to produce different fractions before performing 2DE
Prefractionation
-Subcellular fractionation
-Chromatography
-Immunoprecipitation
2D-DIGE
2D-Difference gel electrophoresis
Features
-Allows running of multiple samples in a single gel
-So no experimental variation
-reduce time
-Quantitation possible with software
Process
-Label with different fluro for each protein extract.
-2 excitation wavelengths produces 2 images, which can be analysed.
Cyanine Dye
-Binds to one lysine per protein, 3 % of all proteins
-Dyes matched for size and charge.
Liquid-phase IEF
-Liquid IEF
-Collect 20 Fractions
-Run each Fraction on Reversed-phase HPLC
-Collect every fraction and run on MALDI-Tof MS to get MWs
-Perform proteolytic digestion on every fraction
-Do MS/MS analyses and get Sequence data
-Identify by Database searching
Affinity-based separations
Retains/binds proteins or peptides based on specific interaction between ligand and ligate
-His tag: immobilized metal affinity-chromatography (IMAC)
-Glycosylated proteins/peptides enriched by lectin affinity chromatography (Concanavalin A, Wheat germ agglutinin, and Jacalin lectin) : GLYCOMICS
-Avidin affinity chromatography to capture biotinylated peptides
2D-Liquid chromatography
-1st dimension: Ion exchange phase
-2nd dimension: Reverse phase
Multidimensional Protein Identification Technology (MudPIT)
Features
-“Shotgun” proteomics
-Whole cell lysates proteolytically digested
-2-dimensional HPLC separation (Cation exchange – reverse phase)
-Fractions are analyzed on-line by ESI MS, or off-line by MALDI-TOF-TOF MS
-Molecular mass, de novo peptide sequencing
-Protein identification based on peptide sequences
Advantages
-Automated procedure
-High throughput
-Identification of
–Lower copy number proteins
–Low and high MW proteins
–Acidic and basic proteins
–Membrane proteins
-But complex MS spectra – computing resource
-Reliability of protein identifications ??
-Validation important
Isotope Coded Affinity Tags (ICAT)
For quantitatiting proteins.
-Separation ICAT label all cysteinyl residues
-Quantitiation: MS to compare ICAT ratios
-Identification with chracterization: MS/MS with database search
Steps
-Proteins from two states (e.g., control versus diseased) are:
–Denatured and reduced
–Labeled at cysteinyl residues with light or heavy ICAT reagents (8 dalton difference)
-Two samples are combined and digested with trypsin
-ICAT-labeled peptides are isolated (captured) by biotin-affinity chromatography
-Analyzed by on-line HPLC coupled to tandem MS
-Ratio of ion intensities for an ICAT-labeled pair quantifies the relative abundance of its parent protein in the original cell state
-Tandem MS reveals the sequence of the peptide and unambiguously identifies the protein
The ICAT technique in action:
Whenever using the ICAT technique to study a proteome, one must have the sample as well as a reference. For example, the sample may be cancerous liver cells and the reference would be normal healthy liver cells.
The proteins from the cancerous cells are labeled with D8 ICAT and the proteins in the healthy cells are labeled with D0 ICAT. The two samples are combined and digested with trypsin, then the ICAT labeled Cys peptides are purified by avidin affinity chromatography. The sample is then run by MS and MS/MS.
The MS data will show two peaks for every protein… one from the D0 ICAT peptide (healthy) and one from the D8 ICAT peptide (cancerous). The difference in height of these two peaks tells us the relative amounts present in each sample. Thus, one can tell if the protein has been up or down regulated in the cancerous cells with respect to the healthy cells (in the example above, the protein in the cancerous cells appears to be present at only half the concentration as in the healthy cells.
The MS/MS data is used to identify the protein by sequence tags (as shown in the earlier slides).
Solid phase isotope tagging
UV induced cleavage at the photocleavable linker significantly reduces background interference
iTRAQ
-isobaric Tags for the Relative and Absolute Quantitation
-4 isobaric reagents enabling quantitation of 4 different samples at one time
-Reagents consist of a reporter -, balance-, and peptide reactive-group (PRG) that labels primary amines (e.g,. Lysine side chains)
-This reagent will tag most tryptic (and free N-terminal) peptides, producing 4 different peaks for the same peptide
Steps
-Mix peptides +iTRAQ MS
-Reporter balance peptide intact MS/MS
-Peptide fragments equal, reporter ions differnet
Applications
-Cellular response to treatment (1 control + three time points of treatment)
-Cancer biomarker discovery (2 normal tissue + two cancerous tissue)
-Enrichment of protein fractions (distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins)
SILAC:Stable Isotope Labeling by Amino Acids in Cell Culture
-Cell lines are grown in media lacking a standard essential amino acid, but
-Supplemented with a non-radioactive, isotopically labeled form of that amino acid E.g., Deuterated leucine (Leu-d3)
-Protein populations from Experimental and Control samples are mixed directly after harvesting
-MS identification is possible as every leucine containing peptide incorporates either normal leucine or Leu-d3
Limitations
-Not applicable for tissues (need to culture cells)
-Stable-isotope-enriched media might affect cell proliferation and possibly protein levels
-Expensive process due to high cost of stable-isotope-enriched media
-Increase in nominal mass is NOT known until sequence is determined
-Well known that isotopic tags bearing deuterated labels separate in reverse phase chromatography
-Other amino acids instead of leucine (e.g., 13C-arginine) have been selected.
13C6-Arginine SILAC
-Tryptic peptides will contain C-termini arginines and lysines
–About ½ of the peptides will contain this label (equal frequency)
–Position of label is thus known
–Mass difference between the labeled and unlabeled is 6
-The change of 12 to 13 in atomic weight of carbon is proportionately much less than the change from 1 to 2 in hydrogen
-Avoids changed chromatographic behaviour.
-Main drawback is high cost , ~ 10-fold higher than Leu-D3.
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