Ultimate 3000 hplc system

PLK1 kinase active mutant T210D was incubated with GST-NUDT1 (2 μg) in kinase buffer for 1 h at 30 °C. The reaction mix was subjected to SDS-PAGE and Coomassie blue staining. Gel bands corresponding to GST-NUDT1 were excised and extracted proteins were digested with trypsin in 50 mM ammonium bicarbonate at 37 °C overnight. After treatment with 5 mM dithiothreitol and 11 mM iodoacetamide, the resulting peptides were separated by a silica capillary column and eluted at a flow rate of 0.3 mL/min using the UltiMate 3000 HPLC system (Thermo Fisher Scientific), coupled with the Q Exactive mass spectrometer (Thermo Fisher Scientific). Data-dependent acquisition mode was adjusted by the Xcalibur 2.2 software. LC–MS/MS analysis was performed in the Protein Chemistry Facility, Center of Biomedical Analysis at Tsinghua University (Beijing, China) according to previous reports^{38 (link)}.

Fifty milligrams of each frozen sample were set aside, transferred to a 1.5-mL Eppendorf (EP) tube (Eppendorf GmbH, Hamburg, Germany), and thawed on ice. The metabolites were extracted with 50% (v/v) methanol buffer-acetonitrile and centrifuged at 4,000 × g for 20 min. The supernatants were stored at -80° C until they were subjected to liquid chromatography-mass spectrometry (LC-MS). The LC-MS was performed in a Thermo Scientific UltiMate 3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a high-resolution tandem Q-Exactive MS (Thermo Fisher Scientific, Waltham, MA, USA) operated in positive and negative ion modes [26 (link)]. An online Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/) annotated the metabolites by matching their exact molecular mass data, names, and formulae with those in the database. A principal component analysis (PCA) was performed to detect outliers in the preprocessed dataset. Differential metabolites had variable influence of projection (VIP) > 1, P < 0.05, and ratio ≥ 2 or ≤ ½, and a KEGG enrichment analysis was performed on them [27 (link)–29 (link)]. Correlations between significant differential genera and metabolites were analyzed by Spearman’s rank correlation test.

Peptide mixtures were dissolved in loading buffer (1% Acetonitrile, 0.1% Trifluoroacetic acid), and 200 fmols/peptide were analyzed by an Ultimate 3000 HPLC system coupled to a high field Q-Exactive (HFX) Orbitrap mass spectrometer (Thermo Scientific). Peptides were trapped by PepMap 100 C18 columns (ThermoFisher Scientific) before reverse phase separation with a 60 min gradient of acetonitrile 2% to 25%, in 1% DMSO, 0.1% Formic acid at a flow rate of 250 nl/min on a 75 μm × 50 cm PepMap RSLC C18 EasySpray column (ThermoFisher Scientific). Data-dependent acquisition involved one full MS1 scan (120,000 resolution, 60 ms accumulation time, AGC 3 × 10⁶) followed by 20 data-dependent MS2 scans (60,000 resolution, 120 ms accumulation time, AGC 5 × 10⁵), with an isolation width of 1.6 m/z and normalized HCD energy of 25%. Three methods were utilized for analysis of the synthetic standard: (A) considered charge states of 2 to 4, (B) considered charge states of 1 to 4 while (C) involved one full scan 300 to 700 followed by 18 MS2 scans for charge states 2 to 4 followed by one full scan 700 to 1400 followed by two MS2 scans for charge states 1. Dynamic exclusion was set for 30 s. For enzymatic digests normalized HCD was increased to 28% and only 2 to 4 charge states were acquired.

1 ml samples of culture medium supernatant were collected either after 4 h and 24 h from 16HBE14 tissue cell cultures with or without Hi2019 infection or from supernatants of bacterial cultures grown to stationary phase. All samples were preserved and prepared for ¹H-NMR analysis as in [34 (link)] in 3 mm NMR tubes. Proton NMR spectroscopy was performed on a Bruker AV900 900MHz spectrometer and one–dimensional proton spectra were measured essentially as in [83 (link)] but using 256 scans. 1D spectra were processed using Topspin 3.0 (Bruker Biospin) and Chenomx NMR Suite 8.2 (Chenomx Inc., Edmonton, Canada) as in [34 (link)]. Detection of acetate, succinate and formate by high-performance liquid chromatography (HPLC) was carried out as in [26 (link)] using supernatants from cultures grown on defined medium with either glucose (10 mM) or lactate (4 mM) and inosine (7.5 mM) as the carbon sources. Metabolite detection used an Ultimate 3000 HPLC system (Thermofisher Scientific) equipped with a UV detector and an RI detector (Shodex RI-101). Samples were separated (total runtime: 20 min) on a Metab-AAC column (300 x 7.8 mm; Isera, Dueren) at 30°C, a flow rate of 0.8 ml/min and a running buffer of 5 mM sulfuric acid. Data analysis used Chromeleon (Thermofisher Scientific).

An Ultimate3000 HPLC system (Thermo Scientific, San Jose, USA) equipped with a quaternary pump, an online degasser, an autosampler, a column temperature compartment, and a UV detector was utilized for LC separation. An XBridgeTMC₁₈ (2.1 mm × 150 mm, 5 μm) was employed at 30°C. The mobile phase consisted of acetonitrile (A) and 10 mmol/L ammonium formate (B; 30 : 70) that was delivered at a flow rate of 0.3 mL/min.
The Q Exactive mass spectrometer (Thermo Scientific, San Jose, USA) equipped with a heat electrospray ionization source operated in the full scan mode was used for MS. The carrier gas was nitrogen, and the pressure of the sheath gas and the auxiliary gas were 35 bar and 10 bar, respectively. Positive and negative ions were scanned simultaneously, and the full scan range was from 150 to 1500 m/z, with the first-order resolution of 70,000. The spray voltage was +3.5 kV or −2.8 kV under the positive or negative mode, respectively. The auxiliary gas heater and capillary temperatures were maintained at 200°C and 350°C, respectively. The ions to be measured and labeled for quantitative analysis were peimine, m/z 432.3472 [M + H]+, peiminine, m/z 430.3316 [M + H]+, and carbamazepine, m/z 237.1022 [M + H]+.

For LC-MS analysis: a 200 μL reaction solution (100 mM HEPES, 150 mM NaCl, pH 7.0) containing 10, or 400 μM of tested samples 4a, (R)- and (S)-4a, and 4g-i, and 100 μg of recombinant ShHTL7 or its mutants was incubated at 25 °C for 30 min. Subsequently, the solution was filtered and analyzed by a nanoACQUITY UPLC system, which was directly interfaced with an SYNAPT-G2-Si mass spectrometer produced by Waters Company. For LC-MS/MS analysis, the above chemicals treated ShHTL7 reaction solution was subjected to the SDS-PAGE analysis. The corresponding protein band around 30 KDa was excised, respectively, then digested with trypsin (Promega) in NH₄HCO₃ solution (50 mM) at 37 °C for 24 h^{23 (link),26 (link)–28 (link)}. The covalent modification of C₅H₅O₂/C₆H₆O on the peptide was analyzed by a Thermo-Dionex Ultimate 3000 HPLC system, which was directly interfaced with a Thermo Orbitrap Fusion Lumos mass spectrometer. MS-MS spectra are generated by pLabel software^{53 (link),54 (link)}.