Ultimate 3000 rslc system

LC-ESI-MS and LC-ESI-MS-MS analyses were done on a LTQ Orbitrap XL ETD mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with standard ESI ion source. 5 μL of sample was injected at a flow rate of 50 μL/min in 80% ACN/H₂O with 0.1% FA by Ultimate 3000 RSLC system from Dionex (Dionex Corporation, Sunnyvale, CA). The conditions for full-scan MS are as follows: mass range m/z 0-6000 and resolution 60,000 at m/z 400. The target ions were sequentially isolated for MS2 by LTQ. Electrospray voltage was maintained at 4 kV and capillary temperature was set at 275 °C.

LC-HR-MS was performed on a Bruker maXis UHR Q-TOFmass spectrometer coupled to an Ultimate 3000 RSLC system (Dionex, Germering, Germany) with a binary pump, an autosampler, a column thermostated at 40 °C, and a DAD detector. A Dionex Acclaim RSLC 120 C18 (250 × 2.1 mm; 2.2 μm) column fitted with a C18 Security Guard Ultra (2.1 mm) (Phenomenex, Le Pecq, France) guard filter was used for LC separations. The mobile phase was water + 0.1% formic acid (solvent A) and acetonitrile + 0.08% formic acid (solvent B). A solvent gradient at a flow rate of 300 μL/min was set as follows: 3% of B for 1 min, then a linear gradient up to 70% of B during 39 min. The column was rinsed with 95% of B for 5 min and then re-equilibrated to the initial conditions for 10 min after each run. 4 μL of sample reconstituted in methanol at 10 mg/mL of engaged material were injected. UV spectra were recorded from 195 to 500 nm. The positive mode using an ESI source in the range of 50 to 1300 m/z at 1 Hz was used to acquire MS data. The parameters were as follows: capillary voltage 4.5 kV, nebulizing gas at 1.5 bar, drying gas heated at 200 °C, at 8 L/min. Data (MS and UV) were acquired with Compass 1.3 software (Bruker) and MS areas were measured with Quant Analysis 2.1 software (Bruker). Extracted ion chromatograms for DPT, PPT, PPTG, MPT and MPTG are presented in Figures S6–S10.

Chromatographic analyses were performed using a Thermo Scientific Ultimate 3000 RSLC system equipped with an autosampler, a binary pump, a thermostated column compartment, a DAD detector (Dionex, Germering, Germany) and an ELSD Sedex 100LT detector (Sedere, Olivet, France) in dynamic gain. The mobile phase was composed of water (A) and acetonitrile (B) with both acidified with 0.1% of formic acid. Elution was performed in the gradient mode at a flow rate of 500 μL·min⁻¹ and with the following binary gradient program: starting with 3% of solvent B during 0.2 min, 3–45%B from 0.2 to 12 min, 45–90%B from 12 to 14 min, and 90–3%B from 15 to 15.5 min. Then, the column was re-equilibrated with 3% of solvent B during 3 min. The column was a Luna^® Omega C18 (150 × 2.1 mm; 1.6 µm) (Phenomenex, Le Pecq, France,) heated at 40 °C and then 10 µL of the oak extracts and fractions were injected.

Desalted tryptic peptides were resuspended in 100 μL of 0.1% (v/v) trifluoroacetic acid. Samples were then analyzed by nLC-MS/MS using an UltiMate 3000 RSLC system (Dionex, USA) coupled to an Orbitrap Elite mass spectrometer (ThermoFisher Scientific, Germany). Initially, peptides were loaded (normalized TIC values between 5 × 10⁸ – 1 × 10⁹, corresponding to 1–4 μL) with 0.1% TFA at 20 μL/min to a 2-cm long (100 μm i.d.) Acclaim® PepMap100 NanoViper Trap column packed with 5 μm silica particles, 100 Å pore size, followed by separation at 250 nL/min on a 50 cm × 75 μm i.d. Acclaim® PepMap100 NanoViper column, both at 60 °C. Peptides were eluted with a gradient of 3 to 45% of 0.1% (v/v) formic acid and 84% (v/v) acetonitrile over 187 min. The spray voltage was set to 1.8 kV with capillary temperature of 275 °C and no sheath or auxiliary gas flow. Full MS spectra were acquired with 1 microscan on the Orbitrap analyzer at a 60,000 resolution (FWHM at m/z 400) with a target AGC value set to 1 × 10⁶. For each survey scan (300 to 1500 m/z range), up to 10 most abundant precursor ions were sequentially submitted to CID fragmentation and MS2 analysis in the LTQ using the following parameters: MSn AGC target value of 1 × 10⁴, normalized collision energy of 35%, minimum signal threshold of 2000 counts and dynamic exclusion time of 30 s.

Three TNBC cell lines (n = 3 per cell type) were lysed in SDS‐lysis buffer. Following protein isolation, disulfide bonds were reduced, and sulfhydryl groups were alkylated with 50 mm Iodoacetamide (IAA) solution for 30 min at room temperature in the dark. After the exchange of buffer with 50 mm triethylammonium bicarbonate (TEAB), the protein was digested at 37 °C overnight using the filter‐aided sample preparation procedure, as previously described [20 (link)], with a trypsin/LysC mixture at a 100 : 1 protein to protease ratio. LC‐MS/MS analysis was conducted for each fraction of the peptide sample using a Q‐exactive plus mass spectrometry (Thermo Fisher) coupled to an Ultimate 3000 RSLC system (Dionex) and a nanoelectrospray source, as previously described, with modifications [21 (link)]. The precursor ions were fragmented with high‐energy collisional dissociation at a normalized collision energy of 32 with a resolution of 35 000 at m/z 200. The maximum ion injection times for the full scan and MS/MS scan were 20 and 100 ms, respectively. The detailed information for LC‐MS/MS analysis can be found in previous studies [14 (link), 21 (link)]. Raw LC‐MS/MS data were uploaded into PRIDE database (PXD005304).

All LC–MS/MS analyses were performed using Quadrupole Orbitrap mass spectrometers, Q-exactive HF-X (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an Ultimate 3000 RSLC system (Dionex, Sunnyvale, CA, USA) via a nanoelectrospray source, as described previously with some modifications^{38 (link),40 (link)}. Peptide samples were separated on a 2-column setup with a trap column (75 µm I.D. × 2 cm, C18 3 µm, 100 Å) and an analytical column (50 µm I.D. × 15 cm, C18 1.9 um, 100 Å). Prior to sample injection, the dried peptide samples were predissolved in solvent A (2% acetonitrile and 0.1% formic acid). After loading the samples onto the Nano LC, a 90-min gradient from 8 to 26% of solvent B (100% acetonitrile and 0.1% formic acid) was applied to all samples. The spray voltage was 2.0 kV in the positive ion mode, and the temperature of the heated capillary was set to 320 °C. MaxQuant.Live version 1.2 was used to perform BoxCar acquisition^{8 (link),41 (link)}. The MS1 resolution was set to 120,000 at m/z 200 for BoxCar, and the acquisition cycle comprised two BoxCar scans at 12 boxes (scaled width, 1 Th overlap) with a maximum ion injection time of 20.8 per box with the individual AGC target set to 250,000.

Samples were analyzed by nano LC-MS/MS analysis using an Orbitrap Fusion Tribrid (Thermo Fisher Scientific, San Jose, CA, USA) mass spectrometer equipped with an “EASY spray” nano ion source (Thermo Fisher Scientific, San Jose, CA, USA). The Orbitrap Fusion Tribrid (Thermo Scientific, San Jose, CA, USA) was interfaced with an UltiMate 3000 RSLC system (Dionex, Sunnyvale, CA, USA). Each sample was reconstituted with 0.1% formic acid in LC-MS-grade water (solvent A; Thermo Scientific, 85178; Rockford, IL, USA), and 5 μL was injected into a nanoviper C₁₈ trap column (3 µm, 75 µm × 2 cm, Dionex) at 3 μL min⁻¹ flow rate, and then separated with a 100 min gradient on an EASY spray C₁₈ RSLC column (2 µm, 75 µm × 25 cm), with a flow rate of 300 nL min⁻¹, and using solvent A and 0.1% formic acid in 90% acetonitrile (solvent B). The gradient was as follows: 10 min solvent A, 7%–20% solvent B for 25 min, 20% solvent B for 15 min, 20%–25% solvent B for 15 min, 25%–95% solvent B for 20 min, and eight min solvent A. The mass spectrometer was operated in positive ion mode with nanospray voltage set at 2.5 kV and source temperature at 280 °C. External calibrants included caffeine, Met-Arg-Phe-Ala (MRFA), and Ultramark 1621 (88323, Thermo Fisher Scientific^TM Pierce^TM; Rockford, IL, USA).