Proteomics Core Services/ Guidelines

Services provided by the Proteomics Core Facility include:

1. 2D gel electrophoresis
2. Protein and peptide mass measurement
3. Protein identification
4. Protein quantitation: proteome-wide (labeled or label-free) and targeted analysis (MRM)
5. Posttranslational modification analysis: phosphorylation, acetylation, etc.

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 and identified with nanLC/nanopray/MS. This novel 2D-DIGE approach can be performed within the Facility. 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.