The following are some general protocols and precautions that ensure a higher success rate.
Sample Handling: (Something to think about: Dogma in Biology - garbage in, garbage out. Dogma in Proteomics - garbage in, MORE garbage out).
The sensitivity of mass spectrometers is both a boon and a bane in the demanding world of biological research. It is a boon because of its ability to characterize vanishingly small amounts of biological materials. The flip side of this advantage is that even small amounts of contaminants can spoil a perfectly planned, well executed experiment. The most common contaminant in the field of protein mass spectrometry is Keratin. Cytokeratin, an abundant protein in skin cell and hair follicles, is everywhere. Since keratins are present as contaminants on the surface of the improperly an handled gel, they get digested more easily than the protein of interest that is inside the gel. Also, keratin peptides ionize more efficiently than the peptides from the protein of interest, thereby, "drowning" their signal. It is almost impossible to avoid keratin, but some "good laboratory practices" can minimize it.
A. Wear gloves all the time and change them often. B. Consider precast gels where applicable. C . Handle the gel as little as possible. D . Keep separate dishes (staining boxes, measuring cylinders, etc.) for gels intended for MS analysis. E. Run a parallel gel for documentation purposes, whenever possible. The surfaces of the light boxes, scanners, etc., are generally not “MS clean”. F . Clean the work area with Methanol or Ethanol.
Differential Protein Expression Analysis by 2D-PAGE
Instrument: For the first dimension IsoElectric Focusing (IEF), PCL uses IPG-Phor system with 18 cm Immobilized pH Gradient (IPG) strip holders. For the 2nd Dimension (SDS-PAGE), Ettan Dalt Six (24cm x 20 cm) gel system is available, which can electrophorese 6 gels at a time.
Reagents: All reagents must be of the highest quality available, preferably PlusOne reagents from Amersham Biosciences (refer to 2D-reagents document in PDF format).
IEF buffers and procedure: Solubilization buffer: PCL has worked with and found the following solubilization buffer to be suitable for most routine IEFs.
Reagents
Concentration
Urea
8M
Thiourea
2M
Tributylphosphine* or DTT
2 mM or 65 mM
CHAPS
4 %
IPG buffer
0.5 %
Tris
40 mM
TBP stock solution (200 mM) is prepared by dropwise addition of 1 ml of TBP to 19 ml of isopropanol. This needs to be prepared freshly as needed.
About 20 ml of solubilization buffer can be prepared and stored at –70 C in 2 ml aliquots for 4-6 months. If using TBP instead of DTT, it might be a good idea to add TBP just before the experiment. Repeated freezing and thawing is not good.
Do not heat the solubilization buffer.
Procedure: Solubilization is the single most important step, which determines the quality of the final separation. Unfortunately, there is no "single magic" solublization protocol available (for detailed discussion about this step please refer to 2D-manual from Amersham Biosciences or 2D protocols in 2D-Swiss database). However, above solubilization buffer is a good starting point. Following is a simple procedure that assumes that solublization is efficient in the solubilization buffer.
Important note:For 18 cm gel format, you will need about 50-100 µg (silver staining) or 300-500 µg (Coomassie staining) total protein in a total volume of 350 µl of the solublization buffer. If the cell/tissue lysis is carried out in a buffer other than the solublization buffer, then the required amount of protein should be in a volume of 35 µl (if not, protein precipitation steps need to be considered).
A. Coomassie stain: Colloidal Coomassie (InVitrogen, LC 6025) is preferred. Homemade Coomassie stains are also acceptable. One problem, however, with the homemade stain is the long and repeated destaining steps involved which might introduce keratin contamination.
B. Silver stain: Mere fact that silver staining is required to visualize the protein of interest indicates the low abundance. Low abundance along with the possibility of keratin contamination renders protein ID after silver staining a very difficult task. Hence the gels should be handled with extreme care. One of the most commonly used method for silver staining of gels for MS purpose is that developed by Vorum. Several MS compatible silver staining procedures are also commercially available.
a. 30 % methanol b. 0.1 M ammonium bicarbonate buffer (no need to adjust the pH) c. One:one mixture of 0.1 M ammonium bicarbonate buffer : Acetonitrile (HPLC grade) d. Reducing buffer: 10 mM DTT in 0.1M ammonium bicarbonate buffer. e. Alkylation buffer: 50 mM Iodoacetamide in 0.1M ammonium bicarbonate. f. Sequencing grade, modified porcine trypsin solution (Promega, V5113). g. 60 % acetonitrile:0.1 % TFA h. Acetronitrile:0.1% TFA. i. Plastic ware: 14 ml falcon tube, 1.5 and 0.5 ml eppendorf tube. All should be pre-washed with acetonitrile and dried.
Procedure: Generally, you need to take less of the gel and more of the protein of interest. Hence, use of 1 mm thick gel is preferred and excise the spot as close to Coomassie stain as possible.
1. Carefully excise the Coomassie stained gel region of interest and place it in a 14 ml Falcon tube. Add 10 ml of 30% methanol and destain it for 3-4 h at RT with constant shaking. 2. Transfer the gel plug into a 1.5 ml eppendorf tube. 3. Add 150-200 µl of Reagent C (Ambic:ACN). Incubate at RT in a Thermomixer, for 30 min. 4. Remove the Reagent C. 5. Add 150 µl of Reagent D (reducing buffer) and incubate for 30 min at RT. 6. Remove Reagent D. 7. Wash once with 150 µl of Reagent C for 2 min. 8. Add 150 µl of Ragent E (alkylation buffer) and incubate for 30 min at RT. 9. Remove Reagent E. 10. Wash twice with 150 µl of Reagent C for 5 min each. 11. Remove Reagent C.
Note:Steps 5-11 are optional but will improve the overall digestion of the protein and recovery of cysteine containing peptides. Might be crucial when maximum coverage of protein (post-translation site identification study) is required.
12. Transfer the gel plug to a 0.5 ml eppendorf tube. 13. Dice/Crush the gel piece into smaller cubes using an eppendorf pestle or a scalpel blade. 14. Dry in Speed Vac for 10 min. 15. Prepare trypsin solution: Dilute 0.5 mg/ml trypsin solution (supplied) to a final concentration of about 25 µg/ml with Reagent B (0.1M Ambic). 16. Add 30 µl (750 ng) of trypsin solution to the dried gel pieces and let the gel swell for 10 min on ice or at RT in trypsin solution. 17. Add 30-50 µl of Reagent B (without trypsin), so that all the gel pieces are neatly submerged.
Note:Dilution of trypsin, and volume of reagent B (without trypsin) added are not fixed. Idea is to add ~600-750 ng of trypsin in minimal volume to the dried gels and allow the trypsin to enter the gels completely to facilitate a proper digestion of protein of interest and minimize the autocatalytic cleavage of trypsin itself. Hence, if the gel plug is small, you may need to do less dilution of trypsin and add less of reagent B in steps 15 and 16 respectively.
18. Incubate at 37 C on the Thermomixer, for 12 h or Overnight. 19. Add another 10 µl (250 ng) of trypsin and continue the incubation for 2 h. 20. Carefully remove the solution (digest) into a clean labeled 0.5 ml eppendorf tube.
Note:Spinning the eppendorf tubes to pellet the gels is NOT useful. Using a 200 µl pipette tip, carefully, avoiding the gel pieces, remove the solution.
21. Add 150 µl of Reagent G (60% ACN:0.1% TFA) to the gel pieces and incubate for 30 min at 30 C on Thermomixer. 22. Carefully remove the solution and pool it with solution from Step 20. 23. Add 150 µl of Ragent I (ACN:0.1 TFA) and incubate for 30 min.
Note: Gel pieces will turn opaque.
24. Carefully remove the solution and pool it with solution from Step 20. 25. Concentrate the digest using a speed vac concentration to a final volume of 15-20 µl. 26. Digest can be stored at -70 C for about 2-4 weeks.
