Q exactive plus orbitrap lc ms ms system

For FT-MS analysis, purified HxlR (at 100 μM) was incubated with 1 mM FA in 20 mM HEPES pH = 7.5, 300 mM NaCl for 1 h at 37 °C. The protein sample was next desalted by Bio-Rad Micro Bio-Spin 6 column and subjected to mass spectrometric analysis by Bruker Solarix XR system. For MS-MS analysis of HxlR protein, 0.6 mM FA was added to the medium of E. coli BL21(DE3) expressing HxlR at 1 h before harvest. The FA-treated cells were then collected and HxlR protein was purified with HisTrap HP column as described above. The purified protein was then digested with trypsin and the resulted peptide sample was next desalted with Thermo Scientific Pierce C18 tips and subjected to Thermo Scientific Q Exactive Plus Orbitrap LC-MS/MS system, with the data collected by Xcalibur software. For LC-MS analysis of synthetic 7-amino acid peptides, the peptides were incubated with 1 mM FA in water for 1 h at 37 °C. The peptide samples were then analyzed with a Waters ACQUITY UPLC I-Class SQD 2 MS spectrometer with electrospray ionization (ESI). Data collection was achieved by Masslynx software.

The labeled peptides were fractionated on a 300 Extend C18 column (4.6 mm × 250 mm, 5 μm, Agilent, USA) and separated with a gradient of 8%–32% acetonitrile (pH 9.0) over 60 min into 60 fractions. The peptides samples were then combined into 14 fractions and dried in a vacuum centrifuge. Subsequently, they were dissolved in 0.1% formic acid solution and loaded onto an EASY-nLC 1000 UPLC system (Thermo, USA). Solvent A was 0.1% formic acid in 90% acetonitrile solution. The gradient comprised an increase from 9% to 26% of solvent B (0.1% formic acid in 90% acetonitrile) over 40 min, a rise from 26% to 35% in 14 min, and then from 35% to 80% in 3 min, followed by holding at 80% for the last 4 min; the flow rate was kept constant at 350 nL/min at all times. The peptides were subjected to NSI source, followed by tandem mass spectrometry (MS/MS) on a Q Exactive Plus Orbitrap LC-MS/MS System (Thermo, USA). The electrospray voltage was 2.0 kV. The mass spectrometer scan range was 350–1800 m/z and the orbitrap resolution was set to 70,000. The MS/MS scan range and orbitrap resolution were 100 m/z and 35,000, respectively. Data-dependent acquisition was used for data collection. Automatic gain control was set to 5E4, and the dynamic exclusion duration was 30 s.

A quadrupole time-of-flight 6600 (AB Sciex) was used for metabolomics data acquisition. Lipidomics data acquisition was performed using a Q-Exactive Plus Orbitrap LC–MS/MS System (Thermo Scientific). The gradients of both separations and the ESI source conditions are described in Supporting Information S2 and S3. Further information on both the separations and ESI source conditions is shown in Additional file 1: S2 and S3.
The metabolomics raw MS data were processed using ProteoWizard and XCMS software. The lipidomics raw MS data were processed using Lipid Search, including peak identification, peak extraction, and lipid identification (secondary identification). Further information is shown in Additional file 1: S4.

The PHGDH-binding proteins in cells were identified by co-IP followed by mass spectrometry (MS) analysis. In brief, HEK293T cells stably expressing FLAG-PHGDH were lysed and subjected to IP using FLAG beads. Immunoprecipitates were separated by SDS-PAGE. The protein bands visualized by Coomassie Brilliant Blue staining were excised and digested in gel with sequencing-grade trypsin (Promega). The digested samples (FLAG-PHGDH-binding proteins) were analyzed on a Q Exactive Plus Orbitrap LC-MS/MS System (ThermoFisher Scientific) and identified using Proteome Discoverer 1.2 software (ThermoFisher Scientific). To identify the potential methylation site for PHGDH, GFP-PHGDH was overexpressed in HEK293T cells and immunoprecipitated with GFP beads, followed by SDS-PAGE and Coomassie Brilliant Blue staining. The GFP-PHGDH protein band was digested in gel by α-lytic protease (MilliporeSigma, A6362), and then analyzed by LC-MS/MS in collaboration with PTM Biolabs (Hangzhou, China).

To verify successful incorporation of acetyllysine, intact proteins were analysed by ESI-MS on a LTQ Orbitrap Discovery mass spectrometer (Thermo Scientific). To validate specificity of the acetylation site, RutR was trypsin-digested and desalted prior to analysis by UPLC-MS/MS. In brief, 4 µg protein were denatured in urea buffer (50 mM TEAB, 8 M urea) and reduced by adding dithiothreitol (DTT) to a final concentration of 5 mM and incubation at 37°C for one hour. Subsequently, proteins were alkylated with chloroacetamide (CAA) (40 mM, 30 min in the dark) followed by LysC digestion for 4 hr at 37°C. Afterwards, tryptic digest was done over night at 37°C. For both enzymes an enzyme to substrate ratio of 1:75 was used. The next day, obtained peptides were acidified by adding formic acid to a final concentration of 1%, desalted and analysed with a Q Exactive Plus Orbitrap LC-MS/MS system (Thermo Scientific) as described earlier (Kuhlmann et al., 2016 (link)). MaxQuant and the implemented Andromeda search engine were used to analyse raw data. MS/MS spectra were correlated with the Uniprot Escherichia coli database containing the target protein (Cox and Mann, 2008 (link); Cox et al., 2011 (link)).