4.6. Proteomic Studies

A total of 1 × 10⁷ cells from each cell line was cultured with or without celastrol, and protein extracts were obtained from homogenization with 100 μL of cold lysis buffer containing 50 mMTris-HCl (pH 7.5), 5 mM EDTA, 10 mM EGTA (ethylene glycol tetraacetic acid), 50 mM NaF, 20 mM KCl, and 250 mM NaCl supplemented with 1 μL of protease inhibitor cocktail. After 1 h at 4 °C, the suspension was frozen and thawed twice in liquid nitrogen. The total extracts were centrifuged at 12,000× g for 30 min, and the supernatants were collected and stored at −80 °C until further processing. Protein concentration was measured by Bradford assay, and the samples were concentrated 39× using a 3 kDa ultrafiltration device (Millipore, Billerica, MA, USA) with 50 mM NH₄HCO₃. In total, 200 μg of protein was used for tryptic digestion, as previously described [52].

Qualitative and quantitative nano-UPLC tandem nano-ESIHDMSE experiments were conducted with a nanoACQUITY UPLC system (Waters, Milford, MA, USA), as previously reported [52]. Briefly, a 180 μm × 23 mm strong cation exchange (SCX) column (Waters) packed with a 5 μm PolySULFOETHYL Aspartamide (PolyLC, Columbia, MD, USA) was used as the first dimension. Nine salt gradient fractions were used to elute the samples from the SCX column, followed by a reverse-phase (RP) gradient. After all of the peptides had been captured, the trap column was placed online with another RP analytical column (100 µm × 100 mm, 1.8 µm C18, nanoACQUITY UPLC HSS T3, Waters, Milford, MA, USA), and an RP gradient of 5–40% acetonitrile (ACN; containing 0.1% v/v formic acid) in 58 min was used as the second dimension, with a flow rate of 600 nL·min⁻¹.

Analyses were performed using nano-electrospray ionization in positive ion mode, nano-ESI (+), with a NanoLockSpray ionization source (Waters, Milford, MA, USA). Multiplexed data-independent (DIA) scanning with added specificity and selectivity of a nonlinear “T-wave” ion mobility (HDMSE; Waters, Milford, MA, USA) experiments was performed with a Synapt HDMS mass spectrometer (Waters, Milford, MA, USA) [52]. Full-scan orthogonal acceleration time-of-flight (oaTOF) MSE data were acquired from m/z 50 to 2000.

Database searching and protein quantification were performed as previously described [52]. Briefly, Exact Mass Retention Time (ERMT) output tables generated for each condition (with or without celastrol) were filtered by considering a well-defined solitary peak without background noise (OK = 2) and differential expression profile (p = 1 up-regulated and p = 0 for downregulated). Additionally, a fold change higher than 50% (with or without celastrol <0.66 or >1.5) was considered to be indicative of significantly altered levels of expression. After filtering, protein data and associated expression profiles were used as input for the curated pathway database Metacore™ software (GeneGO Inc., Saint Joseph, MI, USA).