Sample preparation TMT LC-MS3 mass spectrometry

CELL TREATMENT

H9 hES, Kelly, SK-N-DZ and MM1s cells were treated with DMSO, 1 µM Pomalidomide, 5µM lenalidomide, or 10 µM thalidomide in biological triplicates (DMSO) or biological duplicates (drug) for 5 h and cells were harvested by centrifugation, washed twice with PBS and cell pellets snap frozen in liquid nitrogen.

CELL LYSIS AND PROTEIN PREPARATION

Cells were lysed on ice with the addition of lysis buffer (8M urea, 50 mM NaCl, 50 mM 4- (2hydroxyethyl) piperazineethanesulfonic acid (EPPS) pH 8.5, 1x Roche protease inhibitor and 1x Roche Phosphostop. Cell pellets were homogenized by 20 passes through a 21-gauge (1.25 in. long) needle to achieve a cell lysate which was then clarified by centrifugation at 20,000 xg for 10 min at 4 °C. The clarified cell lysate was transferred to a new tube and a micro-BCA assay (Pierce) was used to determine the final protein concentration. Samples were reduced through the addition of 10 mM TCEP and incubation for 30 min at room temperature. Alkylation was initiated by the addition of 15 mM iodoacetamide and incubated for 45 min at room temperature in the dark, followed by the addition of 10 mM DTT and incubation for 15 min at room temperature in the dark. 200 µg of protein was taken from each sample and moved to a new tube and made up to a total volume of 150 µL with the addition of LCMS grade water.

Proteins were precipitated using methanol/chloroform by addition of 600 µL of LCMS methanol, followed by 150 µL of chloroform and 450 µL of LCMS water. The samples were vortexed to mix and centrifuged at 14,000 xg for 5 min to separate the chloroform phase from the aqueous phase. The aqueous phase was aspirated, and the precipitated protein pellet was then washed with the addition of 450 µL of LCMS grade methanol and centrifuged at 14,000 xg for 5 min to pellet the precipitated protein. The organic layer was aspirated, and the precipitated protein was allowed to air dry.

DIGESTION AND TMT LABELING

Precipitated protein was resuspended in 30 µL of 4 M urea, 50 mM HEPES pH 7.4 followed by dilution of urea to <1 M by the addition of 100 µL of 200 mM EPPS pH 8. 4µg of LysC (ratio of 1:50) was added and digestion allowed to occur for 12 h at room temperature. The samples were then diluted in half to < 0.5 M urea with the addition of 200 mM EPPES pH 8 and samples digested with 8 µg trypsin (ratio of 1:50) for 6 h at 37 °C. Tandem mass tag (TMT) reagents (Thermo Fisher Scientific) were dissolved according to the manufacturer’s instructions – 5 mg of TMT dissolved in 256 µL of anhydrous acetonitrile (ACN). Anhydrous ACN was added to each peptide sample to a final concentration of 30% v/v and mixed, followed by the initiation of labeling with the addition of TMT reagent to each sample at a ratio of 1:4 peptide:TMT label.

The 10-plex labeling reactions were performed for 1.5 h at room temperature and the reaction quenched by the addition of 0.3% hydroxylamine for 15 min at room temperature. 2µL of each of the sample channels was added to 120 µL of 1% formic acid (accurate pipetting required), acidified to pH 2-3 and desalted using C18 solid phase extraction stage tips following the below desalt steps:

Eluted sample was dried in a speed vacuum and resuspended for LCMS submission. Data was analyzed by LC-MS to assess digestion and label efficiency as well as channel ratio comparison. Samples were then combined using the ratio-adjusted volumes determined in the channel ratio analysis and dried down in a speed vacuum. The combined sample was resuspended in 1 mL of 1% formic acid, and acidified (pH 2-3) before being subjected to desalting with C18 SPE (Sep-Pak, Waters):

Eluted sample was dried in a speed vacuum.

REVERSE PHASE FRACTIONATION

Dried sample was resuspended in buffer A and offline fractionated into 96 fractions by high pH reverse-phase HPLC (Agilent LC1260) through an aeris peptide xb-c18 column (phenomenex) with mobile phase A containing 5% acetonitrile and 10 mM NH₄HCO₃ in LC-MS grade H2O, and mobile phase B containing 90% acetonitrile and 10 mM NH₄HCO₃ in LC-MS grade H₂O (both pH 8.0). The 96 resulting fractions were then pooled in a non-contiguous manner into 24 fractions or 48 fractions and dried in a speed vacuum.

96 well plate recombination scheme for 24 fractions:

SAMPLE DESALT

Fractions were resuspended in 100 µL of 5:1 acetonitrile:formic acid, acidified to pH 2-3, and desalted using stage tip C18 solid phase extraction stage tips following the below desalt steps:

Eluted fractions were dried in a speed vacuum and resuspended for LCMS submission. Each fraction was used for subsequent mass spectrometry analysis.