Samples were collected aboard the STING1 cruise aboard the R/V Atlantic Explorer, UNOLS cruise AE2305, in the Gulf of Mexico between the dates of Feb 20 and March 6, 2023. Instruments used were a trace metal clean CTD profiler and a Thermo Orbitrap IQ-X mass spectrometer coupled to a Vanquish Horizon liquid chromatography system.
Sample collection metadata is provided in the Supplemental File section.
Sample collection and solid phase extraction:
Samples were collected from either trace metal clean niskin bottles or deck pump and filtered through 0.2 um membrane filters (polyethersulfone) into acid-cleaned polypropylene bottles to 4L. The sample was then acidified to a pH of 2-3 with hydrochloric acid (trace metal grade) and pumped at 25 mL/min through Bond-Elut PPL solid phase extraction (SPE) columns (1 g, 6 mL, Agilent Technologies) that had been previously activated by passing 6 mL each of methanol (MeOH, Optima LCMS grade, Fisher Scientific) and ultrapure water (qH2O, 18.2 MΩ) through the column. SPE columns were rinsed with 10 mL acidified qH2O (pH = 2) to remove salts and frozen at -20 °C immediately after sample collection and returned to the laboratory for further processing. Sample columns were gravity eluted with 10mL LCMS Optima grade methanol. The eluent was collected in 15mL polypropylene centrifuge tube and evaporated in a SpeedVac to concentrate to a volume of 0.2-0.5 mL. The eluent was transferred to a 2 mL centrifuge tube and brought up to a final volume of 2 mL with qH2O. Each sample was spiked with 20 µL of 100 µM B12 stock for a concentration of 1 µM cyanocobalamin. A pooled sample from each batch of samples for each Orbitrap run was prepared by combining 15 µL from each sample.
Analysis by LCMS:
Chromatographic separation was performed using a Phenyl-Hexyl column (Waters ACQUITY Premier CSH) with dimensions of 2.1 x 100 mm and a 1.7 µm particle size. The column temperature was maintained at 30.0 °C throughout the analytical run. Mobile phase A was composed of water supplemented with 0.1% formic acid, while mobile phase B consisted of methanol with 0.1% formic acid. The system operated at a constant flow rate of 200 µL/min with a 30-min gradient starting from 95% solvent A and 5% solvent B to 5% solvent A and 95% solvent B and a 5 min hold at 95% solvent B. Solvents were then switched back to the initial condition of 95% solvent A and held for 6 min to re-equilibrate the column. The total injection volume for each sample was 25 µL. Mass spectral data were acquired using an Orbitrap IQ-X Tribrid Mass Spectrometer (Thermo Fisher Scientific) operating in positive electrospray ionization (ESI) mode. The ESI source parameters were optimized with a spray voltage of 3500 V, a sheath gas flow of 50 (Arb), an auxiliary gas flow of 10 (Arb), and a sweep gas flow of 1 (Arb). The ion transfer tube and vaporizer temperatures were set to 325 °C and 350 °C, respectively. Full MS scans were recorded in the Orbitrap mass analyzer across a mass range of 100–1000 m/z. Spectra were collected at a resolution of 240,000 at m/z 200.
