Agilent 6210 time of flight mass spectrometer

Cells were collected for metabolome analysis at the indicated infection times. Approximately 2.5 × 10⁶ cells were infected with each V. parahaemolyticus strain at each given time point at an MOI of 50:1. The supernatant was removed, and cells were washed twice with 5 ml of cold 5% mannitol solution. Metabolic activity was rapidly quenched by adding 0.5 ml methanol containing internal standards (100 μM methionine sulfone and camphor 10-sulfonic acid). Intracellular metabolites were extracted using a solvent extraction method by mixing homogenates with 400 μl chloroform and 200 μl Milli-Q water. The mixture was centrifuged (5,000 rpm, 4°C, 5 min). Subsequently, the aqueous layer was filtered using 5-kDa-cutoff filters (Millipore, Bedford, MA) and centrifuged (10,000 rpm, 4°C, 6 h). The filtrate was dried using a vacuum evaporator (4,000 rpm, 4°C, 4 h) and reconstituted in 50 μl Milli-Q water containing spiked internal standards (25 mM [each] 3-aminopyrrolidine and trimesic acid) before analysis.
CE-TOF/MS was performed using an Agilent CE capillary electrophoresis system coupled with an Agilent 6210 time of flight mass spectrometer (Agilent Technologies, Palo Alto, CA) by Human Metabolome Technologies, Inc. (HMT; Tsuruoka, Japan), as described previously (52 (link)).

TBP2 was purified by Strep-Tactin chromatography followed by size exclusion chromatography (Superdex 75; GE Healthcare). Protein purity was determined by Coomassie blue staining of fractions separated by reducing SDS-PAGE. Purified TBP2-StrepII was dialyzed against deionized water 6 times for 2 to 4 h at 4°C by using mini-dialysis tubing (1-kDa molecular-mass cutoff; GE Healthcare). Protein samples were analyzed for accurate mass determination by ESI mass spectrometry. Samples were treated with acetonitrile (50% [vol/vol]) and formic acid (1% [vol/vol]) and immediately loaded into an Agilent 6210 time of flight mass spectrometer (Agilent Technologies, Inc., Santa Clara, CA) with the electrospray ionization source in the positive mode.

The sequences of the purified mAbs and respective F(ab’)₂ and Fc fragments were evaluated by LC-MS using a PoroShell 300SB-C8 column (5 µm, 75 × 1.0 mm) on the Agilent HPLC system followed by analysis in the Agilent 6210 time-of-flight mass spectrometer (Agilent Technologies). The composition of the mobile phase A was 99% water, 1% acetonitrile, and 0.1% formic acid, and that of mobile phase B was 95% acetonitrile, 5% water, and 0.1% formic acid. The gradient started with 20% B at 0 min and increased to 85% B at 10 min with the constant flow rate of 50 µL/min. Each sample was subjected to a native run, a reduced run after incubation with TCEP (Sigma), and a deglycosylated run after incubation with TCEP and PNGase F (New England Biolabs). The MassHunter Qualitative Analysis program (version B.06.00, Agilent, Santa Clara, CA, USA) was used to deconvolute the raw data.

CE-time-of-flight mass spectrometry (TOFMS) was carried out using an Agilent CE Capillary Electrophoresis system equipped with an Agilent 6210 Time of Flight mass spectrometer, Agilent 1100 isocratic HPLC pump, Agilent G1603A CE-MS adapter kit, and Agilent G1607A CE-ESI-MS sprayer kit (Agilent Technologies, Waldbronn, Germany). The systems were controlled by Agilent G2201AA ChemStation software version B.03.01 for CE (Agilent Technologies, Waldbronn, Germany). The metabolites were analyzed by using a fused silica capillary (50 μm i.d. × 80 cm total length), with commercial electrophoresis buffer (solution ID: H3301-1001 for cation analysis and H3302-1021 for anion analysis, Human Metabolome Technologies, Inc., Tsuruoka, Japan) as the electrolyte. The sample was injected at a pressure of 50 mbar for 10 s (approximately 10 nL) in cation analysis and 25 s (approximately 25 nL) in anion analysis. The spectrometer was scanned from m/z 50 to 1,000. Other conditions were as described previously [18 (link)-20 (link)].

Fifty μL of plasma were added to 450 μL of methanol with an internal standard (10 μM final concentration solution of methionine sulfone and 10-camphorsulfonic acid, solution ID: H3304-1002, HMT) at 0 °C in order to inactivate enzymes. The extract was further mixed with 500 μL chloroform and 200 μL Milli-Q water. The mixture was centrifuged at 2300 × g for 5 min at 4 °C, and 350 μL of the upper aqueous layer was transferred into an ultrafiltration tube (Ultra-free MC PLHCC, filter-unit 5 kDa, HMT) and filtered to remove proteins. The filtrate was centrifugally concentrated and rehydrated in 50 μL of Milli-Q water for injection into the CE-TOFMS.
CE-TOFMS measurement was carried out using an Agilent CE Capillary Electrophoresis System equipped with an Agilent 6210 time of flight mass spectrometer, Agilent 1100 isocratic HPLC pump, Agilent G1603A CE-MS adapter kit, and Agilent G1607A CE-ESI-MS sprayer kit (Agilent Technologies, Waldbronn, Germany). The systems were controlled by Agilent G2201AA ChemStation software version B.03.01 for CE (Agilent Technologies). Pre-treated samples were applied into the system using fused silica capillaries (50 μm i.d. ×80 cm total length) (Agilent Technologies) to detect hydrosoluble metabolites. The measurement modes for cation and anion metabolites are shown in Table S1.