Smartprobe

Supernatants were collected, lyophilized and up-concentrated four times in deuterium oxide (Sigma-Aldrich). 1D proton spectra were recorded at 25°C on a Bruker Ascend 400 MHz Avance III HD equipped with a 5 mm Z-gradient SmartProbe (Bruker). The anomeric proton of α-glucose (5.2 ppm), methyl Hβ of lactate (1.3 ppm) and methylene Hγ of glutamine (2.4 ppm) were integrated and quantified by electronic reference to access in vivo concentrations (ERETIC2, Topspin 3.5, Bruker). The methylamine H of a creatine (3.0 ppm) external standard (Sigma-Aldrich) was defined as the ERETIC reference. Consumption/production was normalized to average number of live cells (average of live cell density when treatment was initiated and live cell density at time of harvest) within the 24h time interval examined to obtain consumption/production /cell/24h. Four independent cultures of Um-Uc-3 and T-24 cells were analyzed for each condition.

NMR spectra were acquired
on Bruker Advance III 600 MHz spectrometers with a 5 mm inverse detection
cryoprobe or a 5 mm broadband observe detection SmartProbe, both equipped
with a z gradient. Extracts for NMR were prepared
by treating spider silk (50 mg) with deuterium oxide (D₂O), ethanol-d₆, or DMSO-d₆ in glass vials overnight. Spectra from the D₂O extract were acquired with a 1D NOESY experiment with presaturation
(Bruker pulse sequence noesygppr1d) to suppress the
residual water signal. Spectra were recorded at 25 °C and were
processed with TopSpin 3.6.1. ¹H chemical shifts were referenced
to solvent signals (HDO 4.77 ppm; ethanol-d₆ 1.11 and 3.56 ppm; and DMSO-d₆ 2.50
ppm). Signals were assigned with the help of TOCSY and HSQC experiments.

Each 50 mg of the finely ground leaves, were extracted in MeOD-D₂O (1:1, v/v) as previously described (Kim et al., 2010 (link); Mascellani et al., 2021 (link)). All spectra were recorded on a Bruker Avance III spectrometer equipped with a broad band fluorine observation (BBFO) SmartProbe^™ with z-axis gradients (Bruker BioSpin GmbH, Rheinstetten, Germany), operating at the ¹H NMR frequency of 500.23 MHz. All ¹H NMR spectra were acquired and processed under the same conditions (Mascellani et al., 2021 (link)). All samples were calibrated to the internal standard TMSP at 0.0 ppm, subject to exponential apodization of 0.3 Hz, and phase- and baseline corrections were made using Mnova software, version 14.1.0 (Mestrelab Research, S.L., Santiago de Compostela, Spain).

The ¹H-NMR parameter for spectral acquisition was used according to the protocol by Mascellani et al. (2021) [34 (link)]. All spectra were recorded at 298 K (25 °C) on a Bruker Avance III HD spectrometer equipped with a broadband fluorine observation (BBFO) SmartProbe™ with z-axis gradients (Bruker BioSpin GmbH, Rheinstetten, Germany), operating at a ¹H-NMR frequency of 500.18 MHz. The spectrometer transmitter was locked to deuterated MeOH and all spectra were recorded with the Bruker pulse sequence ‘noesypr1d’ for presaturation of the water signal at 4.704 ppm. Each sample was collected in 64 k data points after 128 scans and 4 dummy scans using a spectral width of 8000 Hz. The receiver gain was set to 18, the relaxation delay was 1 s, the acquisition time was 4 s, and the mixing time was 0.1 s. The free induction decay was multiplied by 0.3 Hz (line broadening) before Fourier transformation. TSP was used to calibrate to 0.0 ppm. The acquired ¹H-NMR spectra were phased and corrected for baseline using Chenomx NMR suite 9 software professional edition (Chenomx Inc., Edmonton, AB, Canada). The assignment of signals was performed using spiked samples, in-house, and built-in databases. The spiking for ergothioneine was performed using the commercially available standard (purity ≥ 98.0%, Sigma-Aldrich, St. Louis, MO, USA) to confirm the annotation.