Dionex ultimate 3000 uhplc system

To identify the phytochemical profiling inside CNSE, the extract was sent to the Institute of Systems Biology (Universiti Kebangsaan Malaysia, Malaysia) for LC-MS analysis using a DionexTM Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Rockford, IL, USA), which coupled with a high-resolution micrOTOF-Q III (Bruker Daltonics, Bremen, Germany). The chromatography was performed on an AcclaimTM Polar Advantage II C18 column (3 mm ⇥ 150 mm, 3 μm particle size) (Thermo Fisher Scientific, Rockford, IL, USA) with a mobile phase containing 0.1% formic acid in water (A) and 100% acetonitrile (B). The gradient conditions consisted of 5% B for 0–3 min; 80% B for 3–10 min; 80% B for 10–15 min; and 5% B for 15–22 min, along with 0.1% A, and a flow rate was set to 400 μL/min. Then, electrospray ionization (ESI) with an ion-positive mode was used as a detector and the m/z values were analyzed by comparing the METLIN (La Jolla, CA, USA) and the KNApSAcK (Keyword Search Web Version 1.000.01) databases, with an accepted error of molecular weight less than 30 parts-per-million (ppm).

The phytochemical profiling of OIE was analyzed using LC-QTOF-MS/MS in negative mode. The analyses were performed on the Dionex Ultimate 3000 UHPLC system (Dionex, USA) coupled with an electrospray ionization (ESI) tandem mass spectrometer (micrOTOF-Q II) (Bruker, Germany). A sample injection volume of 5 μL was used for chromatographic separation of analytes using a Zorbax SB-C18 (250 mm × 4.6 mm × 3.5 μm (Agilent Technologies, USA)) and a gradient program including deionized water containing 0.1% formic acid (FA) as solvent A and acetonitrile containing 0.1% formic acid (FA) as solvent B. The flow rate of the mobile phase was fixed at 0.8 mL/min and the temperature of the column was fixed at 35°C. The gradient program was optimized by passing through the reservoir 30% B, reaching 80% B at 30 min, and holding until 38 min, reducing at 30% B in 2 min, and holding until the run ended at 45 min. The LC-QTOF data were collected and processed by Compass 1.3 software (Bruker, Germany). Apiin, luteolin, quercetin, apigenin, kaempferol, baicalein, and oroxylin A were used as standard reference compounds. The calibration curves were constructed from peak areas of different concentrations of the reference standard (from 1 μg/mL to 100 μg/mL), and the concentrations of targeted compounds were calculated based on the equation for linear regression obtained from the calibration curves.

Quantification of glucose resulting from reactions with the Celluclast® enzyme cocktail was achieved using a Dionex Ultimate 3000 UHPLC system (Dionex, Sunnyvale, CA, USA) equipped with a Rezex ROA-Organic Acid H + (8%), 300 × 7.8 mm analytical column and a SecureGuard Carbo-H + 4 × 3.0 mm guard column (Phenomenex, Torrance, CA, USA) operated at 65 °C. Sample components were eluted isocratically over 22 min using 5 mM sulfuric acid as mobile phase with a flow rate of 0.6 mL/min. Products were detected using a refractive index (RI) detector 101 (Shodex, Tokyo, Japan) and data collection and analysis were carried out with the Chromeleon 7.0 software.

Aliquots (2 mL) of growth medium collected from cultures of P. gingivalis strains ATCC 33277, ∆ppad (ATCC 33277), W83 and Δppad (W83) were lyophilized in an Alpha 1-2 lyophilizer (Martin Christ, Osterode am Harz, Germany) and then dissolved in 100 µL of ultra-pure water to achieve a 20-fold concentration. Three biological replicates were prepared. Samples for electrophoresis were prepared by mixing 15 μL of concentrated supernatant with 15 μL of loading buffer for subsequent SDS-PAGE electrophoresis followed by incubation at 95 °C for 5 min. After electrophoretic separation using the Laemmli system [80 (link)], protein detection was carried out with Coomassie Brilliant Blue G-250 staining and specific protein bands were excised from the gel. These gel samples were subjected to tryptic digestion followed by protein identification with LC-MS/MS using the Dionex UltiMate 3000 UHPLC system (Dionex, Carlsbad, CA) and the HCTUltra ETDII ion-trap mass spectrometer equipped with an electrospray ionization ion source (Bruker, Bremen, Germany), as described previously [81 (link)]. Protein identification was then performed using a SwissProt protein database search (560,118 sequences for all entries, including 336,487 sequences for bacterial proteins) with an in-house Mascot server (v.2.3.0, Matrix Science, London, UK).

Analysis was performed on a Dionex Ultimate 3000 UHPLC system (Dionex, now Thermo Fisher Scientific, Hemel Hempstead, UK) interfaced via an electrospray ionisation (ESI) probe to an Orbitrap Elite MS (Thermo Fisher Scientific). Chromatographic separation was carried out on a Hypersil Gold reversed phase C₁₈ column (1.9 µm particle size, 50 x 2.1 mm, Thermo Fisher Scientific, UK). Details of the mobile phase and gradients employed are given in Supplemental Materials and Methods. MS analysis on the Orbitrap Elite was performed in the positive-ion mode with five scan events, one high resolution (120,000 full width at half maximum height at m/z 400) scan over the m/z range 400 – 610 in the Orbitrap and four MS³ scans performed in parallel in the linear ion trap (LIT). Mass accuracy in the Orbitrap was typically < 5 ppm. More details of the scan events are provided in Supplemental Materials and Methods. Injection volumes were 35 µL for plasma extracts and at 90 µL for amniotic fluid and placental extracts.

The Thermo Scientific Dionex Ultimate 3000 UHPLC system hyphenated with a Thermo Q exactive focus machine used was already reported. For the analysis, 2 mg of each extract were first dissolved in 2 mL of ethanol, then filtered (PTFE filter) and finally 10 μL were injected in the instrument, with all specifications set as previously reported (Salgado et al., 2017 (link); Torres-Benitez et al., 2017 (link)).