Acquity uplc m class

Hydrogen-deuterium exchange was carried out using the commercial Waters HDX system. Briefly: samples were mixed and injected in a labeling time-randomized order by a PAL3 Autosampler followed by UPLC separation and mass spectrometry analysis using a Waters M-Class ACQUITY UPLC with peptides detected on a Waters Select Series Cyclic IMS Mass Spectrometer. Labeling times of 1, 10, and 60 min at RT were used, followed by quenching (7.5 M guanidine hydrochloride and 0.5M TCEP) at 0°C and digestion using an Enzymate BEH Pepsin column. The peptides were reverse-phase separated using an ACQUITY UPLC BEH C18 column. Peptide identification was carried out using HDMSe with CID fragmentation in the transfer cell and ProteinLynx Global Server (PLGS), followed by HDX analysis using DynamX. Structures were visualized using PyMOL 2.5.

The samples for TIMS-TOF analysis were prepared as follows: The agar plugs containing bacteria, confrontation zone, and fungus were immersed in 1 mL of acetonitrile/water/trifluoroacetic acid (70/30/0.1 v/v/v). The mix was stirred at 850 rpm and 20 °C for 5 h (Thermomix.). The supernatant was diluted 1:10 in water before analysis. The chromatographic separation was performed on a M-ClassACQUITY UPLC (Waters, Milford, MA, United States). A 3-min-long sample trapping step was first achieved on a reversed-phase (RP) ACQUITY UPLC M-Class Trap Column (Symmetry C18, 100 Å, 5 μm, 180 μm × 20 mm, Waters, Milford, USA) prior to releasing on a ACQUITY UPLC M-Class BEH C18 analytical column (100 Å, 1.8 μm spherical silica, 75 μm × 100 mm, Waters, United States). Water and acetonitrile both supplemented with 0.1% (v:v) of formic acid (FA) were used as eluents and mixed according to a 32 min-long gradient method. The flow rate was set at 0.6 μL·min⁻¹. The mass detection was performed on a timsTOF spectrometer (Bruker, Bremen, Germany) with the following parameters: timsTOF, scan range m/z, 100–2200. The untargeted profiling data were acquired using Auto MS/MS. Experimental data were processed using Bruker Compass DataAnalysis 6.0.

Peptide separation was performed using an ACQUITY UPLC M-class (Waters). Mobile phase A was 0.1% formic acid in the water, and mobile phase B was 0.1% formic acid in acetonitrile. Samples were reconstituted with 40 µL of SIS peptide mixture in mobile phase A, injected with a full sample loop injection of 5 µL, and separated in an ACQUITY UPLC peptide CSH C18 column (1 mm i.d., 10 cm length, pore size 130 Å, particle size 1.7 µm; Waters). A gradient with a flow rate of 20 µL/min and 5% B for 3 min, 5–25% B for 22 min, 25–60% for 1 min, 60–60% for 1 min, 60–5% B for 1 min, and 5% B for 5 min, followed by column washing with 80% B for 5 min, 80% to 5% for 6 min, 5–80% for 6 min, and re-equilibration with 5% B for 13 min. MRM analysis was performed using a 5500 Qtrap mass spectrometer. The MS detection was carried out in positive mode with the following parameters: ion spray voltage of 5500 V, curtain gas at 20 psi, nebulizer gas at 25 psi, heating gas at 20 psi, resolution at 0.7 Da (unit) for Q1/Q3, interface temperature at 400 °C, and scan mass range 300–1250 m/z QQQ mode. Quantification experiments were performed using the scheduled MRM mode. The MRM detection window time was 120 s, and the cycle time was 1.5 s. The mass spectrometer was operated with Analyst software (Version 1.6.2, SCIEX), which generated MRM-MS data with the file name *.wiff.

Free and bound phenolic compounds were determined using an ACQUITY UPLC M-Class liquid chromatograph from Waters (Waters Corp., Milford, MA, USA) that included a degasser, a binary pump, an oven and an automatic liquid sampler, and it was coupled with mass spectrometry. Phenolic compounds were separated on HPLC column Poroshell 120, SB-C18 (3.0 mm × 100 mm, 2.7 µm) from Agilent Technologies (Agilent Technologies, Palo Alto, CA, USA). Oven temperature was set at 25 °C and separation was conducted according to the conditions previously proposed by Gómez-Caravaca et al. [23 (link)].

To prepare samples for mass spectrometric analysis of phosphorylation site(s) of Sirt1 by LKB1, Sirt1 protein purified from E. coli was incubated with recombinant LKB1-STRADα-Mo25α complex in the presence of ATP and then separated by SDS-PAGE and depicted with colloidal Coomassie blue staining. Following reduction with dithiothreitol and alkylation with iodoacetamide, in-gel digestion of Sirt1 was performed with sequencing-grade Glu-C (Promega, V1651) at 1/50 (Glu-C/protein, w/w) ratio at 37 °C for 16 h in 50 mM NH₄HCO₃ (pH 8.0). The samples were then dried and dissolved in 0.1% formic acid (FA) water. LC-MS analysis was performed on ACQUITY UPLC M-Class (Waters) and XEVO G2-XS QTof Mass Spectrometer System (Waters). The Sirt1 protein samples were separated by a 120-min gradient with the linear gradient from 3% to 80% B (A = 0.1% FA in H₂O, B = 0.1% FA in acetonitrile) at a flow rate of 300 nl/min and the Sirt1-669-peptide samples were separated by a 30-min gradient with the linear gradient from 3% to 80% B (A = 0.1% FA in H₂O, B = 0.1% FA in acetonitrile) at a flow rate of 300 nl/min. The QToF mass spectrometer was operating in MSE mode or target MSMS mode, and the raw data were searched with Progenesis QI for proteomics V4.1 Software against the Human uniprot database.