« back to previous page

Mass Spectrometry Unit

Samples submission: Guidelines for Proteomics sample submission/prep

Additional Information on gels, mass spec, etc.

Gel staining procedures (external link)

• Colloidal coomassie stain protocol
• Silver stain protocol
• Sypro Ruby protocol

Detection limit of proteins from SDS-PAGE gel bands through the use of LC/MS/MS is in the femtomole amounts or on a weight basis in the low nanogram range (1-5ng). This is also very similar to the detection limit of proteins stained with silver (Ag) and Sypro Ruby stains (whereas colloidal coomassie stains typically have detection limits between 10 and 20ng and coomassie Brilliant Blue has a detection limit near 50ng) Detection limits of these stains will be dependent on many variables such as

• gel thickness
• width of the lanes

While the detection limit of protein staining is on a weight basis the detection limit of protein with the mass spectrometer is on a molar basis, therefore the higher the molecular weight, at the same mass, the higher the detection limit will be for the mass spectrometer.
For example:

• 1.0ng of a 20kd protein is 50fm
• 1.0ng of a 200kd protein is only 5fm. Both proteins will have similar stain intensities, but there is 10 times less protein on a molar basis from the 200kd protein.

Protein stains detect total protein, while the mass spectrometer detects proteins individually. Often a gel band contain several proteins, rendering band detectable with Ag stain (i.e. a total of 5.0ng of protein). But there may not be any single protein in the band that would be detectable with the stain by itself and therefore most likely not detectable by the mass spectrometer. For these reasons we encourage the use of colloidal coomassie stain.

Protocols for protein extraction from a cell lysate

To ensure a wide protein coverage for extraction, a tandem extraction protocol can be used, i.e., first use the phenol method to extract one pool of proteins, then the TCA-acetone precipitation on the pellet generated from the phenol extraction to precipitate a different pool of proteins, and finally, combining the pellets of both for analysis.

TCA-Acetone Extraction:
- cells or frozen tissue was added directly to 10% TCA in acetone containing 2% beta-mercaptoethanol (ME) (1 g tissue:10 ml TCA-acetone w/v) and mixed by inverting the tube 10 times
- leave tube at at –20°C for minimum 12 h
- centrifuge precipitated protein at 5000 g for 30 min
- wash three times in 10 mL in ice-cold acetone with vigorous disruption of the pellets with a glass rod between each wash
- air-dry
- to store, freeze pellets at –80°C.

Phenol Extraction:
- make Extraction Buffer:
100 mM potassium chloride (KCl)
0.1 mM PMSF
2% beta-ME
0.7 M sucrose
500 mM Tris
pH 7.5
50 mM EDTA
1% polyvinylpolypyrolidone
1× HALT EDTA-free protease inhibitor cocktail (Pierce, Rockford, IL;
- add cell lysate or tissue (1 g :10 ml buffer w/v) to Extraction Buffer
- add an equal volume of Tris-buffered phenol, pH 7.5
- shake the extraction vigorously on a platform shaker at 4°C for 30 min.
- the extraction was centrifuged at 5000 g and the upper phenol layer removed and re-extracted twice with an equal volume of Extraction Buffer. The final volume of phenol recovered will typically be 1/3 of the starting volume of phenol
- add the final phenol phase to 5 volumes of 0.1 M ammonium acetate dissolved in methanol in order to precipitate the proteins
- tleave this precipitate at –20°C for minimum 12 h
- wash pellets twice in ice-cold methanol, and twice in ice-cold acetone with vigorous disruption of the pellets with a glass rod between each wash
- air-dry
- to store, freeze pellets at –80°C.

Multi-Detergent Extraction:
- make Extraction Buffer:
7 M urea
2 M thiourea
4% CHAPS
2% amidosulfobetaine-14
1% dodecyl maltoside
20% glycerol
200 mM KCl
100 mM dibasic sodium phosphate pH 7.6
1 mM PMSF
- add cell lysate/tissue(1g tissue:10 ml buffer w/v) to Extraction Buffer
- shake extracts moderately for 20 min at room temperature
- centrifuge at 9400 g for 30 min.
- collect supernatant and add it to an equal volume of 10% TCA in acetone containing 2% beta-ME
- leave this overnight at –20°C.
- wash three times in 10 mL in ice-cold acetone with vigorous disruption of the pellets with a glass rod between each wash
- air-dry
- to store, freeze pellets at –80°C.

Tissue Protein Extraction (Bottone protocol, University of North Carolina, Chapel Hill):
Reagents:
Protein extraction buffer
To make 100 mL:
(0.075M Potassium Acetate) 0.736g
(0.3M) NaCl? 1.753g
(0.1M) L-arginine basic salt 1.742g
(0.01M) EDTA-HCl 0.292g
(0.25%) Triton X-100 250. ul
up to 100 ml with dH20, pH 7.4. Then 0.2 um filter.

1. Freeze tissue in liquid nitrogen.
2. Rinse in PBS then mince.
3. Add 1 ml Camiolo extraction buffer per 100 mg of tissue.
4. Homogenize for 1 minute at 4'C.
5. Spin at 3,000. rpm/15 minutes/4'C.
6. Remove supernatant and save in another tube.
7. If necessary, dialize the supernatant against PBS with 50mM/L Tris-HCl pH 7.4.