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 IPGphor IEF System |
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 Ettan Spot Picker |
 Ettan TA Digester |
 Ettan TA Spotter |
 4700 Proteomics Analyzer |
Proteomics Core
Laboratory
Proteomics
The Proteomics Core Laboratory at the Medical College of Georgia offers several services related to protein expression and protein identification. Using a combination of techniques and instruments, this facility is able to take protein samples from several different sources and compare them quantitatively to estimate expression level differences, and ultimately attempt to identify the proteins of interest.
The system utilizes differential dyes from Amersham ® to label the protein samples from different sources. The samples are pooled and then run on the same 2D gel to eliminate as much experimental difference as possible.
Once the gel has been run, it is imaged on a Typhoon imager. Each dye creates its own image, and then the images are compared to quantify relative expression level differences.
Protein spots of interest are identified through certain parameters set in the software. The position of these spots of interested is given a set of coordinates that allow for the Spot Picker to automatically pick the spots and deposit them in a 96-well plate.
This 96-well plate containing the gel plugs is loaded into an automatic tryptic digester. Once the protein has been digested, it is cleaned, dried, re-dissolved and loaded on a slide to undergo mass spectrometry. The spectra created are a “finger print” for the protein. A data base is then searched with the mass spec peak list, and the protein is identified. Some sequence data can be obtained through MS/MS to further enhance the search capabilities.Pre-experimental consideration: Prior to beginning any proteomics experiment, we ask first that an order form be completed and handed to a member of the facility. Second, we request a face-to-face consultation or at least a phone consultation to specify what exactly the investigators overall goal is. We feel it is essential for the investigator to have a clear understanding of what the proteomics facility is and what it has to offer. Optimization for each sample type is almost inevitable due to the special characteristics and diversity of samples and the requirements of individual customers. The following steps can be taken during sample preparation to minimize error.
Sample preparation and submission: Due to the sensitivity of this technology, it is important to take extra precautions in sample preparation. If comparative analyses are desired, it is essential that variables between samples are limited to controlled experimental modifications. We ask that all samples to be used in this manner be prepared the same. In addition, we request that protein samples be cleaned of all possible debris. Each sample for proteomics is to be submitted at a concentration of no less than 50ug of total protein. If an insufficient quantity of protein is loaded, the concentration of the less abundant proteins may be below the sensitivity threshold of the staining system. Also, high protein load helps to increase the possibility of a positive identification during MS. We also ask that the sample is submitted in a buffer that does not contain free amines or free thiols as these will interfere with the CyDye labeling. If desired a quantity of lyses buffer will be provided.
Labeling: The proteomics facility uses Amersham Pharmacia Cy2, Cy3, and Cy5 dyes, both saturating and non-saturating, to label proteins for first and second dimension separation and analysis. The non-saturating dyes label about 3% of each protein, while the saturating dyes label all of the protein.
1-D separation [Isoelectric focusing]: Labeled proteins are next combined with a re-hydration buffer and loaded onto IEF strips of specific pH ranges. Samples are separated by pH range on the IEF strip are then ready for second dimension separation by molecular weight.
2-D separation: The focused proteins are then separated by molecular weight using the standard 2-D PAGE method. The IEF strip is loaded directly into the gel and separation usually takes three hours. The proteomics facility also offers the Ettan DALT II Large Vertical System for simultaneous running of up to twelve large-format gels.
Imaging: The gel is then scanned using wavelengths specific to the particular CyDyes used in the experiment. After initial imaging the gel is then fixed and stained with Sypro Ruby Gel Protein Stain which fluoresces linearly with protein over a range of 10-1000 ng and is compatible with mass spectrometry and amino acid sequencing. Sypro Ruby Gel Protein Stain is comparable in quality and sensitivity to silver stain.
Gel Analysis: Using Decyder Differential In-gel Analysis, the expression-level differences between control and experiment are then determined. The software will then generate a pick list of the interesting proteins from the gel.
Picking: Using the generated pick list, the Ettan Spot Picker will pick the selected spots and transfer them into a 96-well plate.
Digestion: The plate is then transferred to the Ettan TA Digester where an automated trypsin digest is performed. Digested samples are transferred to a clean 96-well plate and dried down completely.
Spotting: Dried plates are next placed onto the Ettan TA Spotter. This machine is designed for high throughput sample spotting on Q-Star, Voyager or 4700 MALDI-TOF target slides.
Mass spectrometry: Instruments used include the ABI 4700 Proteomics Analyzer. Mass spectra are compared against a number of data bases for identification using GPS Explorer.
Data can be sent to the investigator in a number of different ways depending on how much involvement the investigator would like to have in the project. Charges may be adjusted according to customer involvement. For information concerning pricing and access to the facility, please see the “Fees” section.
Tools for data analysis:
MS and MS/MS Search Engines
There are several search engines available for protein identifications,
based on the mass spectrometry analysis. Most often we use the following:
- Mascot Peptide mass fingerprint, sequence query and MS/MS ion search from Matrix Science Ltd., London
- ProFound Protein identification by comparison of a peptide map to NCBI nr database using a Bayesian algorithm, from Rockefeller and NY Universities
- Protein Prospector A variety of tools from UCSF (MS-Fit, MS-Tag, MS-Digest, etc.) for mining sequence databases in conjunction with mass spectrometry experiments
In addition to these there are many other mass spectrum analysis programs and databases you can find on the web site: www.expasy.ch
It is good to calibrate your spectrum before generating a mono-isotopic mass list for peptide mass fingerprinting, e.g. by using trypsin autolysis products as an internal standard. You can find helpful tables on the Search Engines Web Pages.
The 2-DE separation provides us with information on the mw and pI of the protein of interest, so you can use them when evaluating your search result, even though the mass will not be very accurate (you can expect about +/- 10 % difference).
This facility relies heavily on Mass Spec for protein and peptide identification and characterization. Certain Mass Spec techniques may be used outside of the classic proteomics experiments. For more information, please contact the facility. Please click hear for general pricing guidelines (Pricing). For more exact pricing on a project, please contact the facility.