F1.5 thermomixer

After S. cerevisiae was grown in YEPD for 4 h post-dilution, cells were counted in YEPD using Neubauer Improved (NI) hemocytometer (InCyto, Cheonan, Korea, #DHC-N01-2). A total of 700,000 cells were aliquoted into a 1.7 mL microfuge tube and heat shock stimulation was applied by placing the tube in an Eppendorf F1.5 Thermomixer set to 42 °C and 500 rpm for 20 min. At the end of the heat shock incubation, the microfuge tube containing the S. cerevisiae cells were placed on ice for 5 min. The cells were then washed once with ice-cold 1X PBS (Teknova, Hollister, CA, USA, #P0195) and 0.01% BSA (NEB, Ipswich, UK, #B9000Sm), henceforth referred to as PBS-BSA, quickly recounted, and brought to a concentration of 700,000 cells/mL in PBS-BSA. A total of 10 µL of RNase Inhibitor was added to 1 mL of yeast cells in PBS-BSA and mDrop-seq was performed as described below. The emulsion droplets were collected on ice to preserve the heat shock signal during the droplet encapsulation period.

First, 10 μL of fortified serum was diluted into 90 μL of PBS buffer. 59 μL of this 10× diluted solution was spiked into 941 μL of blank human serum. This enriched serum was subsequently used for all reaction assays (Figure 1).
25 μL of serum was added to a 1.7 mL microcentrifuge tube on ice, followed by 8.5 μL of AFB₁-Lys-¹³C₆,¹⁵N₂ internal standard (25 ng/mL). 111.5 μL of buffer (PBS, PBS with 0.45 M Urea, TRIS or ammonium bicarbonate all adjusted to pH 7.5), water and finally Pronase was added. The amount of water was adjusted based on assay conditions so that the final volume was 200 μL in all experiments. The mixture was incubated on a F1.5 thermomixer (Eppendorf, Mississauga, ON, Canada) at 700 rpm at either 37 °C, 50 °C or 60 °C. After incubation, the reaction was quenched by the addition of 200 μL of methanol. 8.5 μL of the second internal standard, AFB₁-Lys-D₄ (25 ng/mL) was then added, the samples were vortexed briefly and centrifuged at 8000 rpm for 10 min at 4 °C. 200 μL of supernatant was transferred to a polypropylene HPLC vial for LC-MS/MS analysis. For assays that performed a protein precipitation step prior to digestion, 200 μL of methanol was added to 25 μL of serum, which was vortexed and centrifuged at 8000 rpm for 10 min at 4 °C. The supernatant was discarded and the pellet was resolublized with PBS buffer or PBS with urea as described above.