Sample Preparation Protocols for Proteomics


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

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            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

 (Source: University of California, San Francisco Mass Spectrometry Facility (http://msf.ucsf.edu/protocols.html)

 CProtocol 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.