Tmt16plex isobaric label reagent

Proteins from inputs and RNase eluates of eRIC were incubated with DTT (10 mM in 50 mM HEPES pH 8.5) for 30 min at 56 °C for the reduction of disulfide bridges in cysteines. Reduced cysteines were alkylated with 2-chloroacetamide (20 mM in 50 mM HEPES pH 8.5) at room temperature for 30 min in the dark. Samples were further processed using the SP3 protocol^{90 (link),91 (link)} and digested with trypsin (sequencing grade, Promega) using an enzyme-to-protein ratio of 1:50 for overnight at 37 °C. Peptides were then recovered by collecting supernatant on a magnet and combining with a second elution wash of beads with HEPES buffer. Subsequently, peptides were labeled with TMT16plex Isobaric Label Reagent (#A44521, Thermo Fischer Scientific) according to the manufacturer’s instructions. Samples were combined for the TMT16plex and further cleaned up using an OASIS® HLB µElution Plate (Waters). Offline high pH reverse phase fractionation was carried out on an Agilent 1200 Infinity high-performance liquid chromatography system, equipped with a Gemini C18 column (3 μm, 110 Å, 100 ×1.0 mm, Phenomenex)^{92 (link)}.

Reduction and alkylation were performed with dithiothreitol (56 °C, 30 min, 10 mM in 50 mM HEPES, pH 8.5) and 2-chloroacetamide (room temperature, in the dark, 30 min, 20 mM in 50 mM HEPES, pH 8.5). Samples were prepared according to the SP3 protocol (10.15252/msb.20145625, 10.1038/s41596-018-0082-x). In short, sequencing-grade trypsin (Promega) was added in an enzyme-to-protein ratio of 1:50 for overnight digestion at 37 °C. Peptide recovery was performed in 50 mM HEPES, pH 8.5 by collecting the supernatant on the magnet and combining it with a second elution.
Peptides were labeled with TMT16plex Isobaric Label Reagent (ThermoFisher) according to the manufacturer’s instructions. In short, 0.8 mg reagent was dissolved in 42 µl acetonitrile (100%) and 8 µl of stock was added and incubated for 1 h at room temperature. The reaction was quenched with 5% hydroxylamine for 15 min at RT. Samples were combined and cleaned up with an OASIS® HLB µElution Plate (Waters).