Briefly...

• Lyse your cells in 0.5-2% sodium deoxycholate (SDC), optionally add benzonase and protease inhibitors

• Determine the protein concentration using BCA (compatible with SDC)

• Add buffer to your sample e.g. TEAB pH 8.5 (pH must stay basic during the protocol otherwise SDC precipitates)

• Reduce disulfide bonds by TCEP (5 mM) at RT-37°C for 60 min

• Block the free –SH groups by IAM (15 mM) at RT for 30 min in the dark

• Quench IAM by cysteine (20 mM) at RT for 30 min in the dark

• Add trypsin 1:50 – 1:100 (w/w) and incubate overnight at 37°C, optionally pre-digest by Lys-C 1:100

• Remove SDC by acid precipitation or phase transfer to ethyl acetate

• Desalt (and optionally delipidate) peptides using SPE on a C-18 cartridge

Thank you so much.

Hi Maynak,

Make sure the pH drops sufficiently during acid precipitation - you might need to verify on a few test-samples using pH-test strips. Look for pH < 4 or so and visible precipitate. Also, SDC and ice do not mix well, depending on your SDC concentration you might get viscous samples when they are kept on ice. 

I actually do not desalt my samples, but configured our HPLC to load onto a trap-column and reverse-elute onto the separating column. Instead of desalting, we spin the sample twice. But then, I do MRM and not SILAC runs.

Good Luck!

Thank you