Spin filter

Cells were lysed in 150 µl lysis buffer (10% SDS, 150 mM NaCl, 100 mM Hepes NaOH pH 7.6) and DNA was sheared by sonication for 5 s. Cell lysates were cleared by centrifugation at 16,000 g for 3 min. 20 µg of total protein were diluted in 4% (w/v) SDS, 100 mM HEPES/NaOH pH 7.6, 150 mM NaCl, 0.1 M DTT and heated at 95°C for 5 min. The lysates were then mixed with 200 µl 8 M urea, 50 mM Tris/HCl, pH 8.5 and loaded onto spin filters with 30 kDa cut-off (Microcon, Merck, Germany). The filter-aided sample preparation protocol (FASP) was essentially followed (Wiśniewski et al., 2009 (link)). Proteins were digested overnight with trypsin (sequencing grade, Promega). According to Kulak et al. (2014 (link)), acidified peptides (0.1% trifluoroacetic acid final concentration) were desalted with C18 StageTips and fractionated with strong cation exchange (SCX) StageTips. The C18 trans-elution fraction was combined with the first of 6 SCX fractions. Peptides were dried and resolved in 1% acetonitrile, 0.1% formic acid.

HyHEL5 and HyHEL10 heavy- and kappa light-chain Fab sequences were synthesized and cloned into the pCEP4 expression vector (Thermo Fisher Scientific) via KpnI and BamHI restriction sites. Heavy chains were C-terminally His-tagged for purification purposes. Fab fragments were transiently expressed using the Expi293 Expression System (Thermo Fisher Scientific) according to the manufacturer’s recommendations using a 1:2 heavy-chain to light-chain ratio. Fab fragments were purified from cell-culture supernatant using HisTrap FF Crude columns (GE Healthcare) according to the manufacturer’s instructions. After dialysis against phosphate-buffered saline (PBS), Fab fragments were concentrated using spin filters (EMD Millipore) and analysed by SDS–PAGE, and their concentrations were determined by spectroscopy (absorbance at 280 nm).

Fifty milliliter SC cultures of SOD2HA C. neoformans at a starting OD₆₀₀ = 0.1 were supplemented with 5 μM CuSO₄ or 1 mM BCS and grown for 16 h at 30 °C, 200 rpm. Cultures were harvested and washed three times in 1X PBS +5 mM EDTA. Cell pellets were resuspended in lysis buffer (10 mM Tris [pH 7.5], 20 mM NaCl, 1 mM EDTA, 1 mM PMSF, and 1X yeast protease inhibitor) and lysed using acid-washed glass beads in a Bead Ruptor 12 (30 s on, 2 min off, ten cycles). Lysates were then pulse sonicated on ice (15 s on, 45 s off, output setting 3, four cycles) and clarified by centrifugation at 20,000g for 15 min 150 μl HA-resin (ThermoFisher Scientific) prewashed in lysis buffer was added to clarified cell extracts and incubated on rotary platform overnight at 4 °C. Resin was pelleted and washed in 3 × 500 μl PBST, followed by another 500 μl PBS. Sod2-HA was eluted using 10 × 100 μl 3 M NaSCN. Elutions were pooled and concentrated using spin filters (EMD Millipore), then buffer exchanged into 10 mM Tris (pH 8.0) using desalting columns (ThermoFisher Scientific). Concentrations were measured using the theoretical extinction coefficient of 40,910 (M⁻¹ cm⁻¹) at A₂₈₀ nm on a Nanodrop One^C (Thermo Scientific) as well as by BCA assay (Pierce). Protein purity was assessed by SDS-PAGE analysis and Coomassie staining.

Isolated extracellular vesicles were delipidated as described previously (10 (link)) using the protein precipitation protocol of Wessel and Flügge (50 (link)). Delipidated extracellular vesicles were resolubilized in 100 μl of 50 mM triethyl ammonium bicarbonate (TEAB) in 1:1 (vol/vol) trifluoroethanol-water. For reduction of cysteine thiols, the solution was mixed with 10 mM Tris(2-carboxyethyl) phosphine and alkylated with 12.5 mM chloroacetamide at 70°C for 30 min in the dark. Proteins were digested for 18 h at 37°C with trypsin-LysC mix (Promega) at a protein-to-protease ratio of 25:1. Tryptic peptides were first completely evaporated using a vacuum concentrator (Eppendorf) and then resolubilized in 0.05% (vol/vol) trifluoroacetic acid (TFA) in 2:98 (vol/vol) acetonitrile-water. Finally, the samples were filtered through Ultrafree-MC hydrophilic polytetrafluoroethylene (PTFE) membrane (0.2-μm pore size) spin filters (Millipore) and stored at −20°C until measurement. Each sample was measured in triplicate (three analytical replicates of three biological replicates) as follows.

Multivalent PEG (40 kDa, eight-arm) containing maleimide-reactive handles (JenKem Technology) was dissolved in 100 mM phosphate buffer (pH 7.0) and filtered (pore size, 0.2 μm). After filtration, the cysteine-terminated peptide–DNA conjugates were added at 2-fold molar excess to the PEG and reacted for at least 4 h at room temperature. Unconjugated molecules were separated using size-exclusion chromatography with a Superdex 200 Increase 10/300 GL column on an ÄKTA fast protein liquid chromatograph (GE Healthcare). The purified nanosensors were concentrated by spin filters (molecular weight cut-off, 10 kDa; Millipore), and quantified with a Quant-iT OliGreen ssDNA Assay Kit (Thermo Fisher Scientific). Fluorescence was read on a Tecan Infinite Pro M200 Quant-iT plate reader at λ_ex = 485 nm, λ_em = 535 nm. Particles were stored at 4 °C in PBS. Dynamic light scattering (Zeta Sizer Nanoseries, Malvern Instruments) was used to characterize the hydrodynamic diameter of nanoparticles. Sequences of DNA-barcoded synthetic urine biomarkers are listed in Supplementary Table 4.

HSA was purified according to a previous method (Curry et al., 1998 (link)). To prepare the HSA complex, we mixed 100 µL HSA (100 mg/mL), 380 µL 2.5 mM palmitic acid (PA), 45 µL Cu(BpT)Br (10 mM), and 45 µL of NAMI-A (10 mM) overnight. Then, the mixtures were concentrated to 100 mg/mL with a Millipore spin filter (10,000 Da cutoff). HSA complex crystals were grown using sitting drop vapor diffusion according to the following procedures (Gou et al., 2015a (link); Qi et al., 2016c (link); Gou et al., 2017 (link)). We mixed 1 µL HSA complex with an equal volume of reservoir solution containing 28–32% (w/v) polyethylene glycol 3350, 50 mM potassium phosphate (pH 7.5), 5% glycerol, and 4% DMSO. HSA complex crystals were frozen in liquid nitrogen when the crystals were picked from solution.
We used the beamline BL17U in the Shanghai Synchrotron Radiation Facility to collect X-ray diffraction data at 100 K (Wang et al., 2015 ). The HSA complex data were integrated and scaled by HKL2000 (Otwinowski & Minor, 1997 ) (Table S1). HSA complex structures were resolved by molecular replacement in CCP4i (Krojer et al., 2017 (link)), and they were refined in the PHENIX program according to the reported procedure (Adams et al., 2010 (link)) (Table S1). The HSA complex structure figures were depicted by PyMOL software (DeLano, 2004 ).