Proteomics and Metabolomics

Sample Preparation Protocols

Metabolomics

Sample Preparation Protocol for cells (adherent and suspension, 10 cm2 plates)

(Note: A sample consisting of ≈ 5 µL of cell pellet, which would require approximately 2.0 x108 cells, would be sufficient to yield successful metabolomics analyses.)

1. For adherent cells, wash plate with 10 mL of medium. Remove medium completely.
2. Incubate for 2 hours in a fresh 10 mL aliquot of medium. Decant excess medium. Aspirate off residual medium.
3. Immediately add 5 mL of Chloroform/Methanol/Water: (1:3:1 ratio; at -80°C), and transfer to -80°C incubator. During the transport of the cell mixture to the -80°C incubator, the contents of cell culture must be submerged, at all times, in a Dry Ice/Ethanol bath, held at -80°C.
4. Incubate mixture for 15 minutes
5. After 15 minutes of incubation, scrap plate with a cell scraper (while still submerged in Dry Ice/Ethanol bath at -80°C).
6. Transfer cell lysate to 15 mL conical tube that is submerged into a Dry Ice/Ethanol bath at -80°C.
7. Centrifuge at full speed for 5 minutes in cold room.
8. Transfer supernatant to a fresh 15 mL conical tube that is submerged in Dry Ice/Ethanol bath at -80°C.
9. To the pellet in the 15 mL conical tubes, add a fresh aliquot of 500 μL of the extraction solvent (Chloroform/Methanol/Water: (1:3:1 ratio; at -80°C)). Re-suspend thoroughly; may require sonication.
10. Transfer mixture to 1.5 mL Eppendorf® tube submerged in Dry Ice/Ethanol bath at -80°C.
11. Spin in micro centrifuge at full speed for 5 minutes in cold room.
12. Transfer supernatant to the 15 mL conical tube that is submerged in Dry Ice/Ethanol bath at -80°C (of step 8).
13. Add 500 μL of the extraction solvent (at -80°C) to the pellet in the 1.5 mL Eppendorf® Re-suspend vigorously.
14. Spin in micro centrifuge at full speed for 5 minutes in cold room
15. Transfer supernatant to the 15 mL conical tubes (in Dry Ice/Ethanol bath at -80°C) (of step 8).
16. Evaporate pooled extracts to dryness using a speedVac or a lyophilizer.
17. Submit dried pellet in 1.5 ml Eppendorf® tube (can be stored at -80°c).

Proteomics

Protocols for trypsin digestion

A. In-solution digestion protocol

Reference: Kinter, M., and Sherman, N. E. (2005) The Preparation of Protein Digests for Mass Spectrometric Sequencing Experiments. in Protein Sequencing and Identification Using Tandem Mass Spectrometry. pp 147-165

Reagents:

1. 1M NH4HCO3 (ABC): 79 mg/mL ultra pure water (fresh, keep on ice)
2. Reducing reagent – 200 mM dithiotreitol (DTT)/100 mM ABC: dissolve 31 mg DTT in 900-µL of ultra pure water and add 100 µL of 1M ABC.
3. Urea solution – 8M urea/100mM ABC/5mM DTT. Place 480 mg urea in tube, add 100 µL of 1M ABC, add 25 µL of 200 mM DTT, and adjust the volume to 1 mL with ultra pure water.
4. Alkylating agent – 200 mM iodoacetamide (IAA)/100 mM ABC: dissolve 37 mg IAA in 900 µL of MS water and add 100 µL of 1 M ABC.
5. Buffer for dilution: 50 mM ABC/2 mM CaCl2.
6. Trypsin solution – 0.2 µg/µL. Prepare just before use. First, 50 mM ABC/2 mM CaCl2, keep on ice. Add 100 µL of ice-cold 50 mM ABC/2 mM CaCl2 to 20 µg of sequencing-grade modified trypsin (Promega). Dissolve trypsin by drawing the solution into and out of the pipette. Keep this solution in ice until use.

Digestion:

The protein sample (we used pellet after Amersham clean-up) is evaporated and re-suspended in urea solution (i.e. with DTT; solution #3 above) up to the protein concentration ~ 5 µg/µL. Mix carefully by drawing the sample into and out of the pipette.

1. Reduction: 1-2 hour at room temperature or 1 hr at 30ºC in a mixer. Adjust to room temperature and spin down.
2. Alkylation: Add alkylating reagent (200mM IAA) up to 15 mM to the reaction mixture. Incubate 45 minutes at room temperature in the dark.
3. Add the same amount of the reducing agent to consume any un-reacted IAA. Incubate ~ 20 minutes (Example: add 1.5 µL of 200 mM DTT).
4. Reduce the urea concentration up to 1 M by diluting the reaction mixture with 50 mM ABC/2 mM CaCl2 (Example: add 140 µL of 50 mM ABC/CaCl2 to 20 µL of starting sample solution).
5. Add trypsin solution to the reaction mixture at 1:30 – 1:40 ratio. Vortex gently, put for digestion overnight (16 – 18 hr) at 37 ºC (Example: if a total protein amount – 100 µg, add 12.5 µL of 0.2 µg/µL trypsin solution to achieve 1:40 ratio).
6. Next Day: Stop reaction by adding concentrated (10%) TFA or FA and adjust pH to ~ 5.
7. Add also acetonitrile (ACN) up to 2% if you plan to desalt the digest by micro, Macro trap desalting cartridges (see Michrom guide to Trap Cartridge care and use). Test the pH by placing the 1-L aliquots of the samples onto an appropriate pH paper.
8. Desalted digest can be analyzed by 2D LC/MS/MS. For 1D LC/MS – (if you have peptide Trap in-line to wash out reagents) you can use un-desalted digest. Keep unused digest at -20ºC.

 

B. In-Gel Digestion Protocol

University of California, San Francisco Mass Spectrometry Facility

C. Protocol for immunoprecipitation

Teng, Y., Ngoka, L., Mei, Y., Lesoon, L., and Cowell, J. K. (2012) HSP90 and HSP70 Proteins Are Essential for Stabilization and Activation of WASF3 Metastasis-promoting Protein. The Journal of biological chemistry 287, 10051-10059.