Monday, March 9, 2015

Efficient sample preparation protocol for analysis of cellular proteome by LC-MS/MS

Winter and Steen (2011) have tested various cell lysis, protein precipitation, and digestion protocols and reported as Technical Brief in Proteomics journal (link to the paper). They showed that the best method of sample preparation for LC-MS/MS analysis of HeLa S3 cells is syringe lysis of cells in GlyNP40 buffer, chloroform/methanol precipitation of the extracted proteins, and in-solution digestion supplemented with a surfactant.

The protocol covers three stages of sample preparation:  
  1. Cell Lysis
  2. Protein precipitation, and 
  3. Enzymatic digestion
1. Cell Lysis:

GlyNP40 lysis buffer composition


150 mM KCl

1mM MgCl2

10% Glycerol

0.5% NP-40


Protease inhibitors

  • Harvest the cells grown on 15 cm culture dishes.
  • Resuspend the cell pellets in 500 µl of GlyNP40 buffer.
  • Syringe lysis - Pass the cell suspension five times through a 26.5 Gauge needle. 
  • Centrifuge the sample for 30 min at 20,000 g at 4°C.
  • Collect the supernatant.
  • Perform protein assay of the supernatant. Divide the sample in 100 µg aliquots and store at -80 °C or proceed to the next step for protein precipitation. 
Cell lysis efficiency of GlyNP40 and RIPA buffer are comparable; however, RIPA buffer contains SDS that may precipitate with proteins during chloroform/methanol precipitation.

Composition of RIPA buffer: 25 mM Tris/HCl, 150 mM NaCl, 1% NP40, 1% SDS, 1 mM EGTA + Protease inhibitors

2. Protein precipitation:
Use aliquots containing 100 µg proteins for chloroform/methanol precipitation.  
  •  Prepare ice-cold solution of chloroform and methanol mixed in 2:1 ratio. Mix it in a 5:1 ratio with the cell lysates.
  • Vortex for 20s. Centrifuge at 20,000 g for 30 min at 4 °C. After centrifugation you should see separate phase of chloroform (bottom) and methanol/water (top) with a layer of precipitated proteins in between the two phases. 
  •  Use a Hamilton syringe and carefully remove first the bottom chloroform phase and then the top methanol/water phase.
  • Add 1 mL ice-cold methanol to wash the protein pellet. Centrifuge at 20,000 g for 15 min at 4 °C. 
  •  Remove the supernatant and air dry the pellets.

Chloroform/methanol precipitation has many advantages: it is fast; it precipitates at neutral pH, which is beneficial for the study of labile modifications; and it forms pellet with a large surface area, which are easy to solubilize for enzymatic digestion.  

3. Enzymatic digestion:

  • Resolubilize the pellets by adding 30 µl of 0.1% RapiGest (prepared in 100 mM triethyl-ammonium bicarbonate (TEAB) buffer) and incubating for 45 min at 37 °C.
  • Reduce with DTT (final concentration 5 mM) for 45 - 60  min at 56 °C.
  • Alkylate with iodoacetamide (final concentration 15 mM) for 30 – 45 min at room temperature in the dark. 
  • Adjust the final volume of the sample to 100 µl using 100 mM TEAB.
  • Add 1 µg trypsin (enzyme/protein ratio = 1/100) and incubate overnight at 37 °C.
  • Next day, add 5 µl of TFA (5% final concentration) and incubate at 37 °C, 800 rpm in a thermomixer for 45 min.
  • Centrifuge at 20,000 g for 30 min. This step removes RapiGest from the solution. 
  • Transfer the supernatant to a new tube. The sample is ready for analysis by LC-MS/MS. 

The protocol was used and optimized for the analysis of HeLa S3 cells, but I don’t see why it should not work for other mammalian cell types.
Winter D, Steen H. Optimization of cell lysis and protein digestion protocols for the analysis of HeLa S3 cells by LC-MS/MS. Proteomics 2011 Dec;11(24): 4726-30