Q exactive ms

The peptide mixture of each sample was separated on a high-performance liquid chromatography (HPLC) system (EASY-nLC 1000, Thermo Finnigan, San Jose, CA, USA). After HPLC separation, the peptides from all replicates were analysed using a Q-Exactive MS (Thermo Finnigan) for 120 min^{40 (link),41 (link)}. Notably, each sample was processed three times, and the MS experiments for each sample were performed in triplicate to avoid contingency of the date and assure data reliability. The liquid chromatographic conditions, elution gradient, and Q-Exactive MS requirements are described in Supplementary S-3.

Total proteins extraction from rice leaves was performed using a PEG-mediated prefractionation method [30 (link)], and protein was digested with trypsin following the FASP method [49 (link)]. The trypsin-digested peptide mixture was then loaded onto aliquot of titanium dioxide (TiO₂) beads (5 μm Titansphere, GL Sciences, Japan), which were then collected by centrifugation after washing twice with 30 mg/mL DHB (2,5-dihydroxybenzoic acid) buffer. The beads were further washed for twice with 60% ACN/0.1% TFA and 0.1% TFA respectively, before elution with a 60% ACN/4% ammonium solution. NanoLC-MS/MS was performed with a Q Exactive MS (Thermo Finnigan) equipped with Easy nLC1000 (ThermoFisher, San Jose, CA). The peptide mixture was seperated on a C18-reversed phase column with a flow rate of 250 nL/min over 240 min. Peptides were analyzed by MS/MS in positive ion mode, and the MS/MS spectra search was performed against the Uniprot_Oryza database (v.2018.02.27), using Mascot 2.2 engine. Proteome Discoverer 1.3 (Thermo Electron, San Jose, CA) was used for identification of phosphorylation peptides, with the threshold setting as pRS score above 50 indicating a good PSM (Peptide Spectrum Matches) and pRS probabilities above 75% indicating a truly phosphorylated site.

LC-MS/MS was performed with an Easy nLC (Thermo Fisher Scientific, Waltham, MA, USA) coupled to Q Exactive MS (Thermo Finnigan, San Jose, CA, USA) [69 (link)]. Briefly, the iTRAQ-labeled peptides (5 μg) were separated by a Thermo Scientific Easy C₁₈ column (75 μm × 100 mm, 3 μm) with gradient elution from 2% B to 45% B in 120 min (A: 0.1% formic acid in H₂O; B: 0.1% formic acid in acetonitrile) at a flow rate of 300 nL/min. All tandem MS were produced following the higher collision energy dissociation (HCD) method. Specifically, MS survey scans were acquired using a data-dependent top 10 method, in which the most abundant precursor ions between 350 and 1500 m/z were dynamically chosen for higher collision energy dissociation (HCD) fragmentation. The resolution was set to 60,000 at 400 m/z, the automatic gain control (AGC) target value was 1 × 10⁶, and the maximum ion accumulation time was 200 ms.

Metabolomics profiling was performed by Ultimate 3000 LC combined with Q Exactive MS (Thermo Fisher Scientific, CA, United States) as reported in our recent publication (Aleidi et al., 2021 (link)). Briefly, extracted metabolites were first separated using an ACQUITY UPLC HSS T3 (100 × 2.1 mm 1.8 μm) and a mobile phase composed of solvent A (0.05% formic acid-water) and solvent B (ACN) with a gradient elution over 16 min applied at 300 μl/min flow rate. MS spectra were acquired in full MS scan in the range m/z 50–1,500, with 25,000 enhanced mass resolution and a frequency 15 spectra per second. The capillary voltage was 3000 and 3200 V for positive and negative ionization modes, respectively. The fragmentation was achieved for MSMS experiments at 175 V, with nebulizer gas at 35 bsi, and gas temperature 450°C. Chromatographic and MS parameters (under positive and negative ionization modes) were kept as reported previously (Aleidi et al., 2021 (link)). Pooled samples prepared the quality control (QC) sample. A QC injection was performed every 10 LC-MS sample runs. In total, there were 18 QC samples injected and analyzed.

Sucralose and acesulfame-K concentrations in plasma, urine, feces and breast milk were measured in triplicate using liquid chromatography-mass spectrometry (LC-MS). Analyses were performed with an Acquity I-Class UPLC (Waters Corp., Milford, MA, United States) and an Acquity UPLC BEH C-18 column (2.1 mm × 50 mm, 1.7 μm) coupled with a Q-Exactive MS (Thermo Scientific, Waltham, MA, United States) with a HESI-II electrospray source. Analyses were performed using isotopically labeled sucralose as the internal standard for sucralose measurements and isotopically labeled acesulfame-K for acesulfame-K measurements. The %CV for the samples throughout experiments was 4.5% for sucralose and 0.8% for acesulfame-K, respectively, and for samples between days was 5.8% for sucralose and 1.0% for acesulfame-K, respectively.