Tmt10

We performed the PISA assay with biological triplicates to identify LNP’s target in C. auris, as previously described [30 (link)]. Briefly, C. auris AR0390 cultures were grown to the logarithmic phase in YPD and were subsequently collected, washed, and adjusted to 1 × 10⁸ CFU/mL in PBS. Cells were treated with either LNP (64 µg/mL) or DMSO. Samples were frozen in liquid nitrogen. Four freezing and thawing cycles were applied to obtain cellular lysates in the presence of a protease inhibitor cocktail. For PISA experiments, the lysates were then heated to eight different temperatures between 40.3 and 60.7 °C (40.3, 44.3, 48.3, 50.3, 52.1, 54.8, 58.3, and 60.7 °C) for 3 mins followed by incubation at 25 °C for 5 mins. The samples were ultracentrifuged at 150,000×g for 30 min at 4 °C, and the supernatant was collected. Subsequently, the soluble protein solutions were then precipitated, reconstituted in 8 M Urea, reduced/alkylated, digested using Trypsin/Lys-C, and labelled with TMT10 (Thermo Fisher Scientific Inc, MA, USA) as per vendor protocols for subsequent high-pH fractions and quantitative LC–MS/MS proteomics analysis. In the case of the global proteomics experiments, the same sample preparation procedure for LC-MS/MS was executed for 30 µg of total protein, excluding heat treatment and mixing. The detailed methods were adaptations from the previously reported [31 (link)].

Proteins from each sample were processed by Filter aided proteome preparation (FASP) method [19 (link)] was used for trypsin digestion, the filtrate was collected, and the peptide was quantified (OD280). Tandem mass tags TMT10 (Thermo Fisher Scientific, USA) with varying molecular weights (126–131 Da) were used as isobaric tags for relative and absolute quantification. According to manufacturer's protocols, the digested samples were individually labeled with TMT10 reagents for 1 h as follows: 100 μg of aliquots of digested peptides of the Control group, Model group, or JWDSD-H group were each labeled with a different isobaric tag (TMT126, 127, 128, 129, 130 and 131, respectively). The labeling reaction was stopped with 5% hydroxylamine. As directed, the Pierce high pH reversed-phase fractionation kit (Thermo scientific) was then used to fractionate TMT-labeled digest samples into 10 fractions using an increasing acetonitrile step-gradient elution.

Tumor samples for MS-proteomics were prepared as previously described^{35 (link)}. The protein fraction from the Allprep kit (Qiagen) was prepared for mass spectrometry-based proteomics using a modified version of the filter assisted sample preparation method (FASP)^{80 (link)}. Samples were mixed with 1 mM DTT, 8 M urea, 25 mM HEPES, pH 7.6 in a centrifugation filtering unit, 10 kDa cutoff (Nanosep® Centrifugal Devices with Omega™ Membrane, 10 k), and centrifuged for 15 min at 14.000 g, followed by another addition of the 8 M urea buffer and centrifugation. Proteins were alkylated by 55 mM IAA, in 8 M urea, 25 mM HEPES, pH 7.6 for 10 min, centrifuged, followed by two more additions and centrifugations with 8 M urea, 25 mM HEPES pH 7.6. Trypsin (Promega). 1:50, Trypsin:protein, was added to the samples in 0.250 M urea, 25 mM HEPES and digested overnight at 37 °C. The filter units were centrifuged for 15 min at 14.000 g, followed by another centrifugation with MQ. Flow-through of peptides was collected and TMT10 labeled according to manufacturer’s instructions (Thermo). A TMT tag with pool of all samples was used as denominator in each TMT10 to connect the different sets. TMT labeled peptides were pooled and cleaned by a strata-X-C-cartridge (Phenomenex).