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| Proteomics |
These services are provided by the Proteomics Core Laboratory:
Differential Protein Expression Analysis by 2D-PAGE: (Currently UT/BGSU users only)
This technique, pioneered by O'Farrell et al. (J. Biol. chemi. 1975, 250, 4007-4021), separates proteins based both on their isoelectric point (pI) and molecular mass (Mr) resulting in far better resolution of proteins compared to one dimensional gel separation methods. During early days, the technique was bogged down by the difficulty in getting reproducible 2D maps partially due to the problems associated with casting and handling of isoelectric focusing (IEF) tube gels. In the 1990's, commercial development of Immobilized pH Gradients (IPG) and advances in proteomic technologies rekindled the interest in 2D-PAGE.
A detailed procedure is available in General Protocols. Protein samples (e.g., cell, tisse lysates from control and treated groups) are denatured and solubilised using a combination of denaturants, detergents and suitable ampholytes, and applied onto an IPG strip. Under an electric field, proteins start migrating toward their respective pls. Once they reach their pl, net charge on the protein equals zero and proteins stop migrating. These IEF gels are then layered onto the 2nd dimension SDS-PAGE for separation based on the Mr. Proteins are visualized using a suitable stain (Coomassie, silver, or fluorescence-based stains). Routinely about 2,000-3,000 proteins can be resolved on an 18cm gel format.
Limitations of Differential Protein Expression Analysis by 2D-PAGE |
1. Compared to several hundred thousand proteins present in many experimental models, resolving capability of 2D gels is limited. However, availability of several narrow pf range IPG strips has partially alleviated this problem.
2. Under-representation of membrane proteins, and of basic proteins.
3. The gel is only as good as the staining technique employed. Even though fluorescent and silver staining techniques are sensitive to low ng level, several proteins of interest (such as regulatory proteins) might be below the detection limit. In such cases, enrichment of the sample, using conventional separation techniques will be needed.
In spite of these and other limitations, 2D-PAGE is still one of the best methods available for differential protein expression analysis.
For more information:
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. Limitations of Protein Identification by Peptide Mass Mapping:
1. Technical limitations: Instrument can routinely obtain data from as little as one pmole of the peptide mixture. However, salts and detergents interfere and may prevent good quality data from being obtained. Most often, peptides obtained by in-gel digestion are desalted/cleaned up using Ziptip C18 reverse phase pipette tips (Milipore) prior to spotting on the MALDI target. Generally, peptide/protein should be dissolved in a volatile solvent (water, acetonitrile, etc.) at a concentration of at least 1 pmole/µl.
2. Complexity of the sample: Since protein identification is based on the probability of experimentally-determined peptide masses (NOT actual peptide sequences), matching that of a protein in the database, the experimental protein should be relatively pure (ex. aspot from 2D-PAGE). If the sample contains more than 2-3 proteins, MALDI analysis often results in ambiguous results.
3. Database requirement: Availability of the appropriate, annotated database is a pre-requisite for successful identification of proteins using this MALDI technique. |
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Protein Identification by Peptide Sequencing using Liquid Chromatography - Tandem Mass Spectrometer (LC-tandem MS): In this technique, mass and sequence (using mass of daughter ions) of a parent peptide is determined in two sequential MS scans, hence the name tandem-MS. Peptides are ionized at atmospheric pressure and introduced into the in-let of the mass spectrometer. In the first MS scan, these peptide ions are guided into an ion-trap where the ions are stored and sequentially scanned out and mass determined (parent peptide masses). In the second MS scan or tandem-MS or MS/MS, a single parent peptide (per experimental set up) is trapped while throwing out other peptides. This trapped peptide collides with a dampening gas (He) in the ion-trap, which results in breaking of the peptide, mostly at peptide bonds, generating daughter ions (Collision Induced Dissociation, CID). Like the first MS scan, these daughter ions are sequentially scanned out and masses determined resulting in a CID spectrum. From the masses of parent and daughter ion, the sequence of the peptide is determined by comparison with known sequences in the database (if available) or deduced by manual interpretation (de novo sequencing).
Peptide in a peptide mixture (either from a single protein or multiple proteins as is often the case with protein complexes) are resolved using an online reverse phase HPLC column. Our facility uses Aquasil C18 Picofrit columns (5 cm x 75 µm i.d. x 15 µm tip, NewObjective) to achieve a reliable and high degree of peptide separation. Peptides are directly introduced into the inlet of the mass spectrometer using ThermoFinnigan’s nano-spray ionization set up.
Since the peptides are separated using a reverse phase column and are identified based on actual sequence information, tandem-MS is well suited for analysis of complex mixtures of proteins and/or proteins isolated from organisms whose annotated genome database is not available.
Currently PCL is accepting Coomassie stained (preferably Colloidal Coomassie stain, InVitrogen) gel spots/bands for analysis by either MALDI or tandem MS. Electrophoretic technique removes salts and other contaminants from the sample thereby enhancing the success of mass spectrometry identification. Silver/Sypro ruby stained gel spots/bands are also acceptable. However as the amount of proteins in such samples are low (1-10 ng), and inherent inefficiency of in-gel digestion method (60-80 % recovery), success rate drops down considerably (>50%). Using modified silver staining method (see General Protocol) and handling the samples extremely carefully, increases the success rate.
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