From 20 to 25 µg of proteins (based on two lowest temperatures in case TPP experiments, and NP40-processed vehicle-treated samples in case of SPP experiments) from the supernatant were reduced, alkylated, and digested with Trypsin and LysC using modified SP3 protocol50 (link). Briefly, proteins samples were bound to acidified paramagnetic beads (0.5 µg Sera-Mag-beads A and B in 10 µl of 15% formic acid and 30 µl ethanol). Following an incubation of 15 min, the protein-bound beads were washed four times with 70% ethanol. On bead reduction, alkylation, and digestion of the proteins were performed overnight using 0.25 µg Trypsin, 0.25 µg LysC, 1.7 mM TCEP, and 5 mM chloroacetamide in 100 mM HEPES, pH 8. Subsequently, the peptides were eluted from the beads and lyophilized. The dried peptides were dissolved with 10 µl water and 10 µl of TMT labels (8 µg per µl) was added and incubated for 60 min. The labelling reactions were quenched with 5 µl hydroxylamine (2.5%) solution and pooled together and vacuum dried for LC-MS-MS analysis.
For TPP-temperature range experiments, sample from 10 different temperatures were labelled with 10 different TMT tags and pooled together as a single TMT experiment. In a 2D-TPP experiment, the concentration range of two neighboring temperatures were labelled and pooled as a single TMT experiment. In case of SPP experiment, nine NP40-processed (one vehicle and eight small molecule treated) samples and one SDS-processed (vehicle) sample were labelled and pooled as a single TMT experiment.
Dried peptide samples were reconstituted in 1.25% ammonia in water and fractionated on an Ultimate3000 (Dionex) by reversed-phase chromatography at pH 12 (Buffer A: 20 mM Ammonium formate pH 10, Buffer B: Acetonitrile) on X-bridge column (2.1 × 10 mm, C18, 3.5 µm, Waters, Milford, MA), and 24 fractions were collected and vacuum dried.
For TPP-temperature range experiments, sample from 10 different temperatures were labelled with 10 different TMT tags and pooled together as a single TMT experiment. In a 2D-TPP experiment, the concentration range of two neighboring temperatures were labelled and pooled as a single TMT experiment. In case of SPP experiment, nine NP40-processed (one vehicle and eight small molecule treated) samples and one SDS-processed (vehicle) sample were labelled and pooled as a single TMT experiment.
Dried peptide samples were reconstituted in 1.25% ammonia in water and fractionated on an Ultimate3000 (Dionex) by reversed-phase chromatography at pH 12 (Buffer A: 20 mM Ammonium formate pH 10, Buffer B: Acetonitrile) on X-bridge column (2.1 × 10 mm, C18, 3.5 µm, Waters, Milford, MA), and 24 fractions were collected and vacuum dried.
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