Sample Preparation for LC-MS/MS

Protein extraction: 3 x 106 MDA-MB-231 cells, treated with L42 or vehicle control, were collected via scraping, centrifuged and then washed twice with ice-cold 1X PBS. Samples were centrifuged at 3000 rpm for 5 min after each wash, and pelleted cells were re-suspended in 100 �l of 100 mM ammonium bicarbonate (ABC). Samples were then homogenized at 35000 psi using a Barocycler (PBS, Inc) with high-pressure time of 20 s followed by low-pressure time of 10 s, for each cycle for a total of 90 cycles. Homogenized samples were centrifuged at 14000 rpm for 20 min at 4 �C and the soluble fraction (SF, supernatant) and membrane-fraction (MF, pellet) were collected separately. SF was subjected to bicinchoninic acid (BCA) assay to measure protein concentration. A total of 100 �g of SF was made up to 100 �l using Milli Q water and then precipitated using 5 volumes of cold (-20  �C) acetone overnight to allow for protein precipitation. For pellet (membrane fraction, MF), 10 �l of urea/DTT was added and MF samples were placed in 37 �C rotary shaker for 1 h to reduce and denature proteins. Denatured MF samples were then mixed with 10 �l alkylation mixture (195 �l acetonitrile (ACN), 1 �l triethylphosphine, and 4 �l 2-iodoethanol) and placed for a further 1 h in 37 �C shaker. MF samples were then dried in vacuum centrifuge for 1 h after which the dry protein pellets were stored at -20 �C until further use. Precipitated SF samples were centrifuged at 14000 rpm for 20 min and pellets were dried in vacuum centrifuge briefly, after which SF samples were also subjected to reduction, denaturation and acetylation as described previously.

Trypsinization and solid phase extraction (SPE):  Lys-C/Trypsin vial containing 20 �g of the mixture was dissolved by adding 400 �l of 25 mM ABC, and 80 �l was added to each sample (both SF and MF) to achieve enzyme:substrate ratio of 1:25. Samples were then transferred to a barocycler for digestion at high pressure. Proteins were digested for 1 h at 50�C at 20 kpsi using a program set at high pressure for 50s followed by a low pressure for 10s for each cycle, for a total of 60 cycles. Digested peptides were desalted using C18 SPE columns using a protocol provided by the manufacturer. Clean peptides were eluted using 50 �l 80% ACN/ 0.1% FA/ 20% Milli Q, and elution step was repeated twice. Eluates were dried in a SpeedVac (???), following which the clean, dry peptides were stored at -20 �C until further use. 

LC-MS/MS analysis: Samples were analyzed by reverse-phase HPLC-ESI-MS/MS using the Dionex UltiMate 3000 RSLC nano System (Thermo Fisher Scientific) coupled to the Q-Exactive High Field (HF) Hybrid Quadrupole Orbitrap MS (Thermo Fisher Scientific) and a Nano- electrospray Flex ion source (Thermo Fisher Scientific). Peptides were re-suspended in 3% ACN/0.1% FA/97% milliQ formic acid, and loaded onto a trap column (300 ?m ID ? 5 mm) packed with 5 ?m 100 � PepMap C18 medium and washed using a flow rate of 5 �l /minute with 98% purified water/2% acetonitrile (ACN)/0.01% formic acid (FA). The trap column was then switched in-line with the analytical column after 5 minutes. Peptides were separated using a reverse phase Acclaim PepMap RSLC C18 (75 �m x 15 cm) analytical column using a 120-min method at a flow rate of 300 nl/minute. The analytical column was packed with 2 ?m 100 � PepMap C18 medium (Thermo Fisher Scientific). Mobile phase A consisted of 0.01% FA in water and a mobile phase B consisted of 0.01 % FA in 80% ACN.  The linear gradient started at 5% B and reached 30% B in 80 minutes, 45% B in 91 minutes, and 100% B in 93 minutes. The column was held at 100% B for the next 5 minutes before being brought back to 5% B and held for 20 minutes to equilibrate the column. Sample was injected into the QE HF through the Nanospray Flex� Ion Source fitted with an emission tip from Thermo Scientific. Column temperature was maintained at 35?C. MS data were acquired with a Top20 data-dependent MS/MS scan method. The full scan MS spectra were collected over 300-1,650 m/z range with a maximum injection time of 100 milliseconds, a resolution of 120,000 at 200 m/z, spray voltage of 2 and AGC target of 1 �106. Fragmentation of precursor ions was performed by high-energy C-trap dissociation (HCD) with the normalized collision energy of 27 eV. MS/MS scans were acquired at a resolution of 15,000 at m/z 200. The dynamic exclusion was set at 20 s to avoid repeated scanning of identical peptides.  Instrument optimization and recalibration was carried out at the start of each batch run using the Pierce calibration solution. The sensitivity of the instrument was also monitored using an E. coli digest at the start of the sample run.