Protein digestion

For cell line samples’ phosphoproteome analysis, aliquots of cell lysates were digested in a StageTip using our recently developed streamlined approach modified from Kulak et al.⁴⁶. Briefly, the StageTip was prepared by packing three layers of reverse-phase SDB-RPS Empore^TM disks in 1 mL pipette tips. Protein samples extracted in lysis buffer cocktail containing reducing and alkylating agents from cells were loaded into StageTip and digested for 16 h with Lys-C 1:100 (w:w, Lys-C:protein) and trypsin 1:50 (w:w, trypsin:protein). The digest sample is acidified with 0.5% TFA and then five volume of ethyl acetate was added and vortexed (500 × g, 2 min, room temperature). The resultant peptides were washed first with 1:1 ethyl acetate:0.2%TFA (v:v) and then by 0.2% TFA centrifuging (1000 × g, 1 min, room temperature). Peptides were then eluted by 80% ACN in 5% NH₄OH basic solution for single shot or in a stepwise varying % ACN for fractionation.

Methanol/chloroform protein precipitation was performed on the protein extracted from tissue samples¹⁰. For one volume of sample, four volumes of methanol, one volume of chloroform, and three volumes of ultra-pure water were sequentially added with intensive mixing. After centrifugation (10 min at room temperature, at 16,000 × g), the upper aqueous phase was removed without disturbing the interface and the precipitate was washed by three volumes of methanol with thorough vortexing. The sample was centrifuged (5 min at room temperature, at 16,000 × g) and the supernatant was removed. The white protein precipitate was then allowed to air dry. The extracted proteins were resuspended in 8 M Urea, then reduced by 10 mM DTT at 29 °C for 30 min, and alkylated with 50 mM IAM at 29 °C for 30 min in the dark with temperature selection aimed at reducing urea carbamylation^10,47. The samples were diluted with onefold volume of 50 mM TEABC and Lys-C was added at a ratio of 1:100 (w:w, Lys-C:protein) for digestion at 29 °C for 3 h. The samples were further diluted with threefold volume of 50 mM TEABC for trypsin digestion with a ratio of 1:50 (w:w, trypsin:protein) for 18 h at 29 °C. The proteolytic digestion was quenched by adding 10% TFA to a final concentration of 0.5% in the sample. The resultant peptides were desalted by reversed-phase StageTips²². Briefly, SDB-XC (Styrene Divinylbenzene) membrane was packed into the 200 μL stage tip, activated with 80% ACN, and conditioned with 0.5% ACN. After the sample loading, washing was performed under 0.5% ACN. Finally, the peptides were eluted by 80% ACN and transferred into a new tube. The yeast protein extract was also digested in solution and desalted in StageTip.