Proteomics Core Facility

The mission of the Proteomics Facility is to provide and disseminate state-of-the-art scientific and technical knowledge in proteomics that will contribute to the research programs of investigators at the University of Pennsylvania and the Children's Hospital of Philadelphia.

This will be accomplished by exploiting and evaluating the impacts of discoveries in proteomics, through focused efforts in:

1. instrumentation
2. protein separation and detection
3. bioinformatics

The Facility is an integral component of the Genomics Institute.

The Proteomics Facility Office
853 BRB II/III
The Proteomics Facility
838 BRB II/III
Philadelphia, PA 19104-6160
215.573.9883 Tel 215.573.9889 Fax

Chao-Xing Yuan, Ph.D. 852 BRB II/III
Christine Busch 838 BRB II/III
Data Processing Room Proteomics Facility 851 BRB II/III

Services provided by the Proteomics Core Facility include:

1. 2D gel electrophoresis
2. Ettan DIGE analysis
3. Applied Biosystems Voyager Pro DE MALDI-TOF mass spectrometer for peptide mass measurement
4. LCQ Deca XP Plus mass spectrometer for peptide sequencing

2D-ETTAN-DIGE (DIFFERENTIAL IN GEL ELECTROPHORESIS)

The Facility provides a novel approach for quantitative protein expression profiling and protein identification. The 2D-Ettan-DIGE approach for comparative proteomics involves labeling (CyDye DIGE fluors); co-separating extracts from different cell conditions on a single 2D gel, staining (Sypro Ruby) and imaging the gel using sophisticated analysis software (DeCyder). After analysis, spots of interest are picked, the protein within the gel plug is digested with trypsin, the sample is spotted onto a MALDI (matrix-assisted laser desorption/ionization) plate, and a peptide mass fingerprint is determined using a MALDI mass spectrometer. A peptide mass fingerprint has an approximately 50 % chance to identify a protein in a given spot. Unidentified proteins can be analyzed using the LCQ Deca XP Plus mass spectrometer, which provides peptide-sequencing capability. Peptide mass fingerprinting using MALDI and peptide sequencing using the LCQ are complementary. This novel 2D-DIGE approach can be performed within the Facility. The Facility has access to three different fluorescent Cy dyes (Cy2, Cy3 and Cy5) through the Technology Access Program from Amersham Biosciences. With these dyes, up to three protein extracts can be covalently labeled prior to electrophoresis, and an equal amount of each extract is run within the same gel (Tonge, R. et al., 2001 Proteomics 1:377-396).

This approach eliminates gel-to-gel variation, making the analysis less complicated. In addition, fewer replicates are needed to reach a given level of significance, accelerating the data collection process.

THE FACILITY METHOD FOR RUNNING 2D GELS

• First Dimension - Isoelectric focusing (IEF) using immobilized pH gradients (IPG) separates proteins according to their difference in isoelectric points. It is important that proteins are completely solubilized before focusing is started. Otherwise, partially solubilized proteins will inevitably appear at additional spots in the gel. To the solubilized sample, an appropriate amount of rehydration buffer is added. The rehydration buffer consists of 8M urea, 2% CHAPS, a trace of bromophenol blue, 0.4 mg DTT (freshly added) and 0.5% IPG buffer. The suspension is allowed to sit at room temperature for 30 min. The sample is centrifuged to remove insoluble debris. The soluble fraction is applied into the strip holder of appropriate length. An IPG dry strip is added to the solution (strip gel side should be face down). Any air bubbles that may be trapped beneath the strip are removed. Strip rehydration is allowed to take place for 10 hours. Electro focusing is carried out for 45000 to 65000 volt hours, with a maximum of 8000V, depending on strip length and the presence of salts and detergent in the original sample.
• Preperation of Focusing Strip for the Second Dimension - At the end of the first dimensional separation (focusing) the strips are equilibrated for 10-15 min in equilibrating buffer (50mM Tris-HCl, pH 8.8, 6M urea, 30% glycerol, 20mM DTT), followed by 10-15 min equilibration in the above buffer, substituting 4% iodoacetamide for the DTT.
• Second Dimension - SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate the focused proteins according to their difference in molecular weight. The percentage of acrylamide in the separation gel is chosen based on the size of the proteins to be separated. This step takes 1-5 hours depending on the size of gel used. After the separation gels are stained and scanned.

GUIDELINES OF SAMPLE PREPARATION FOR 2-D ELECTROPHORESIS

• General - Samples should be lysed before submission. Precipitated samples must be solubilized in lysis solution and clarified by centrifugation if necessary. The solution or supernatant will be analyzed. Contaminants (see list below) should be removed from the sample prior to submission. Protein concentration must be specified; Samples for DIGE must be 5mg/ml or greater. Each gel will require 50ug of each sample (up to 3 samples may be run on the same gel).

Contaminants
Not Acceptable:		Acceptable:
Reducing agents (e.g. DTT, TBP)	Buffers other than Tris	Urea
Salts (e.g. NaCl)	Ionic detergents (e.g. SDS)	Water
Nucleic acids	Polysaccharides	Tris base
Lipids	Phenolic compounds	Non-ionic or zwitterionic
Insoluble material		detergents (e.g. CHAPS)
Other small ionic molecules

• Lysis Solution - 8 M Urea, CHAPS 2-4% (w/v), 40mM Tris. Protease inhibitor if necessary, e.g. complete mini (Roche) 1 tablet/10 ml buffer. Add protease inhibitor immediately after or during cell lysis
  
• General Hints for Sample Preparation
1. Be sure not to include DTT or any other reducing agents in the sample as they interfere with the labeling of the protein with Cy dyes. A reducing agent will be added after the labeling, but before IEF.
2. In case protein extraction contains components not compatible with 2D electrophoresis, remove these components prior to submission e.g. by precipitating and re-dissolving in lysis solution.
3. SDS containing samples must be diluted to a final concentration of 0.25% SDS or less. Adjust the protein concentration accordingly.
   
• Precautions When Using the LCQ Deca XP Plus for Identification of Proteins in Gel Bands - The LCQ Deca XP Plus mass spectrometer provides a capability of peptide sequencing which gives structural information of the peptides from a given protein or a protein band. Turbo-Sequest software uses the structural information of your peptides to identify the protein of your interest. 1pmol of protein is required for this analysis. Always wear gloves (powderless, rinsed with water and ethanol before use) to prevent contaminations such as keratin. Use clean supplies such as tips, tubes, and scalpels. Use the highest quality reagents (including H2O) available.


• Precautions Before Running the Gel - Optimum thickness for gels is 1 mm. It is desirable to use the narrowest gel lane width possible to achieve protein to gel volume ratio as high as possible.
1. Commercial gels are available such as from Invitrogen.
2. Homemade gels: Polymerize acrylamide gels overnight to minimize cross-linking of unpolymerized acrylamide to proteins during electrophoresis.
   
• Precautions After Running the Gel - Use clean dishes and freshly made staining solutions for staining the gels to prevent contaminations from keratin, dust, saliva, any other proteins you are using in your lab. Please be sure to follow the Coomassie Brilliant Blue Staining Protocol because many staining protocols, although perhaps slightly more sensitive or convenient than this one, may well render your proteins impervious to MS analysis. In general, chemical crosslinkers and strong oxidizing or reducing agents should be avoided. After staining, gels may be stored in 1 to 2% acetic acid.

  When you acquire an image of your gel, please take special care not to allow your gel to contact any contaminated surfaces during the process. When you cut out the bands of interest, be sure to use extremely clean surfaces and new scalpels for band excision. Ideally this should be done in a laminar flow hood (tissue culture type) to minimize contamination from dust, hair, skin flakes, dirt etc. Even trace amounts of such contaminants usually contain keratins in much larger amounts than the proteins present in the gel bands of interest. Therefore, such contaminants can cause the failure of attempts to characterize the proteins. Once cut, gel bands can be stored frozen in water or 1% acetic acid in clean, sealed sample tubes. Blank bands from the same gel are very helpful for measuring the background

Coomassie Brilliant Blue Staining Protocol (for mini-gels):

1. Fix gel in 100 ml 46 % methanol, 7 % acetic acid for 1 hour.
2. Stain gel in 100 ml 46 % methanol, 7 % acetic acid, 0.1% Coomassie Brilliant Blue R-250 (filter this before use) for 1 hour.
3. De-stain gel in100 ml 5% methanol, 7.5% acetic acid for 24 hours. Replace if needed.
4. Store the gel in 1 to 2% acetic acid in clean sealed sample tubes at 4 degrees C.

CHARGES

Item code	Description	Typical Cost
130	2D Large Format Gel (26 x 20 cm), 1-6 gels	$180
135	2D Large Format Gel (26 x 20 cm), >7 gels	$155
136	2D Small Format Gel (8 x 10 cm), per gel	$100
160	SYPRO Ruby Staining and Scanning, per gel	$50
165	Cy2,3,5 Dye labeling and Scanning, per gel	$220
300	Full service image analysis, per hour	$60
400	Spot picking, per 96-well plate	$40
500	Trypsin Digest, 1-10, per sample	$10
505	Trypsin Digest, 11-20, per sample	$8
506	Trypsin Digest, >20, per sample	$5
610	MALDI-TOF Peptide Mapping, 1-10, per sample	$50
611	MALDI-TOF Peptide Mapping, 11-20, per sample	$40
612	MALDI-TOF Peptide Mapping, 21-50, per sample	$30
613	MALDI-TOF Peptide Mapping, >50, per sample	$20
650	Microflow RP-HPLC/MS/MS, 1-10, per sample	$250
651	Microflow RP-HPLC/MS/MS, 11-20, per sample	$150
652	Microflow RP-HPLC/MS/MS, 21-50, per sample	$100
653	Microflow RP-HPLC/MS/MS, >50, per sample	$80
700	Mass Spectrometry Analysis/Database Search, per hour	$60
	15% discount for Cancer Center, Pharmacology and Institute of Genomics

To complete this process, download the following Excel files and save them to your hard drive.

1. New_Sample_form_for_Mass_Spec.xls
2. New_Sample_form_for_2D_gel.xls
3. New_Request_Instrument_Usage_Form.xls

Then follow this link, https://somapps.med.upenn.edu/bmcrc/submission_prot.php and upload your files.