Q exactivetm hybrid quadrupole orbitrap mass spectrometer

Briefly, BAs in 100 mg of fecal samples were extracted using 1 mL of methanol. BAs in the optioned supernatant after 30 min of centrifugation at 12,000 rpm were analyzed. BAs were separated using a Dionex™ UltiMate™ 3000 Rapid Separation LC (RSLC) system (Thermo Scientific, Waltham, MA, USA) equipped with an HSS T3C18 column (2.1 mm × 100 mm, 1.8 μm, Waters, Milford, CT, USA). The injection volume, temperature, and flow rate were set to 50 °C, 10 μL, and 300 μL/min, respectively. Methanol (A) and 2 mmol/L of ammonium acetate (B) were used as the mobile phases. Furthermore, the mass spectrometry was performed using a Thermo Scientific TM Q Exactive TM hybrid quadrupole Orbitrap mass spectrometer equipped with a HESI-II probe. The negative HESI-II spray voltage was 3.5 kV. The heated capillary temperature, sheath gas pressure, auxiliary gas setting, and heated vaporizer temperature were 320 °C, 30 psi, 10 psi, and 300 °C, respectively. The auxiliary gas, sheath gas, and collision gas were all nitrogen at 1.5 m Torr. The full mass scan parameters were set as follows: an auto gain control target under 1 × 10⁶, a resolution of 70,000, an m/z range of 150–1500, and a maximum isolation time of 50 ms [19 (link)].

The proteomic analysis was performed to identify proteins in nectars at the Iowa State University protein facility⁵. The nectar of different species was initially analyzed by SDS-PAGE according method described previously (Laemmli, 1970 (link)). Afterwards, the selected bands were excised and the pieces transferred to a 0.6 mL methanol, washed and then added 20 μl of 1% acetic acid. Next, the proteins were digested in solution with trypsin/Lys-C (Promega). The peptides were then separated by liquid chromatography and analyzed by MS/MS by fragmenting each peptide on Q Exactive^TM Hybrid Quadrupole-Orbitrap Mass Spectrometer from Thermo Scientific. Raw data were analyzed using Thermo Scientific’s Proteome Discoverer Software and the data searched using publically available databases. Bovine serum albumin was used as an internal calibration standard.

The labeled peptides were dissolved in 0.1% FA (Formic acid) and directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Fisher Scientific). Peptide separation was performed in a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific). The gradient was comprised of an increase from 6% to 22% of solvent B (0.1% FA in 98% acetonitrile) over 26 min, then 22% to 35% in 8 min and increasing to 80% in 3 min, then holding at 80% for the last 3 min, at a constant flow rate of 350 nl min⁻¹ on an EASY-nLC 1000 UPLC system. The resulting peptides were analyzed on a Q ExactiveTM hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific).

Comparative analysis of fermentation extracts of all strains containing pOE484 except K7N3, CPN4 and CPN5, relative to control mutant containing empty plasmid pIB139, was conducted at ETH Zurich on a Dionex Ultimate 3000 HPLC system coupled to a Thermo Scientific^TM Q Exactive^TM Hybrid Quadrupole-Orbitrap mass spectrometer. MS-settings: spray voltage 3.5 kV; capillary temperature 320 °C; sheath gas (52.50), auxiliary gas (13.75), sweep gas (2.75); probe heater 437.50 °C; S-Lens RF (50), positive mode, resolution 70.000; AGC target 1e6, microscans 1, maximum IT 75 ms, scan range 200–1800 m/z. Chromatographic separation was obtained using a Phenomenex Kinetex 2.6 µm XB-C18 150 × 4.6 mm column with solvents (A, H₂O + 0.1% formic acid) and (B, MeCN + 0.1% formic acid) and the following gradient: flow rate 0.7 mL min⁻¹, 20% B for 2 min, 20–98% B over 18 min, 98% B for 5 min, 98–20% B in 0.5 min and 20% B for 4 min. Metabolic differences within the obtained data (Supplementary Table 8) were identified using SIEVE 2.0 screening software (Thermo Fischer Scientific), applying the default settings for component extraction of small molecules, except that of the base peak minimum intensity, which was set to 5000000.

The samples were fractionated by high pH reverse-phase High Performance Liquid Chromatography (HPLC). Briefly, peptides were dried by vacuum centrifugation. Then the Peptides were dissolved in 0.1% formic acid and directly loaded onto a reversed-phase precolumn (Acclaim PepMap 100, Thermo Scientific). Peptides were separated using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific) and analyzed by Q Exactive^TM hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific).
The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q Exactive^TM (Thermo Fisher Scientific) coupled online to the HPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using NCE setting as 32. Ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 2E4 in the MS survey scan with 30.0 s dynamic exclusion. The electrospray voltage was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap. 5E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800. Fixed first mass was set as 100 m/z.

Peptides were dissolved in 0.1% FA (Fluka), and loaded directly onto a reversed-phase precolumn (Acclaim PepMap 100, Thermo, Waltham, MA, USA). A Q Exactive^TM hybrid quadrupole-Orbitrap mass spectrometer (Thermo, Waltham, MA, USA) was used to analyze the resulting peptides. The peptides with a treatment of NanoSpray Ionization (NSI) source was analyzed by MS/MS in Q Exactive^TM (Thermo, Waltham, MA, USA). At a resolution of 70,000, complete peptides were detected in Orbitrap. For MS/MS, peptides were picked up by a normalized collision energy (NCE) setting of 28. At a resolution of 17,500, Orbitrap was used to detect the ion fragments. For the top 20 precursor ions above the threshold ion count of 5E3, a data-dependent process was performed between 1 MS scan and subsequent 20 MS/MS scans alternately. The electrospray voltage applied was 2.0 kV. To prevent Orbitrap from overfilling, the automatic gain control (AGC) was used; 5E4 ions were cumulated for the formation of MS/MS spectrum. The m/z scanning ranged from 350 to 1800. The fixed first mass setting is 100 m/z.

Peptides were dissolved in 0.1% FA (Sigma-Aldrich), directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific). The resulting peptides were analyzed by Q Exactive^TM hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific). LC-MS/MS analysis, database search and bioinformatics annotation were performed by PTM-Biolabs Co., Ltd (Hang Zhou, China). Gene Ontology and KEGG pathway analysis and visualization were performed using R (Version 3.5.0) with the package clusterProfiler (Version 3.8.1) 15 (link). Protein-protein interaction networks were predicted by STRING. Network visualization was performed by Cytoscape (Version 3.4.0). Genes were considered to be significantly differentially expressed between groups when the P-value was less than 0.05 and the fold change of expression was more than 2.0.