DOI: 10.5061/dryad.d51c5b087
title: Data From: Shedding light on cobalamin photodegradation in the ocean

description: Cobalamin, vitamin B12, is an important micronutrient that has been
 investigated for decades in the marine context because it is required for
 phytoplankton growth. The biologically active forms (Me-B12, Ado-B12) and
 the synthetic form (CN-B12) quickly convert to OH-B12 after light exposure
 in various aqueous solutions, but puzzlingly have been frequently reported
 to dominate dissolved cobalamin pools in the sunlit ocean. Here we
 document photodegradation timescales for these cobalamin forms in natural
 seawater using targeted mass spectrometry, providing quantitative evidence
 that OH-B12 is expected to be the dominant dissolved form in irradiated
 seawater. Then, through high resolution mass spectrometry, we identify
 four photodegradation products of OH-B12 which represent potential
 building blocks microbes could salvage and remodel to satisfy cellular
 cobalamin requirements. Taken together, these results clarify the impact
 of light on marine cobalamin dynamics, laying a foundation for a more
 quantitative understanding of the role of cobalamin in microbial
 communities and biogeochemical cycles.
description: Analytical cobalamin standards Cobalamin
 standards CN-B<sub>12 </sub>(≥ 98%, Fisher BioReagents),
 Me-B<sub>12 </sub>(≥ 97%, Sigma-Aldrich),
 Ado-B<sub>12 </sub>(≥ 97%, Sigma-Aldrich) and
 OH-B<sub>12 </sub>(≥ 95%, Supelco) were obtained and primary
 stock solutions were prepared by dissolving 1 mg of each compound in 1 mL
 of Optima LC-MS grade water and stored at –80°C in the dark until
 use. Seawater collection We
 collected seawater from CTD rosette bottles into amber HDPE bottles while
 protecting from light, then filtered (0.2 µm pore-size nylon filters)
 while protected from light and stored at -20°C in acid-washed, milli-Q
 water rinsed, and sample-rinsed amber HDPE bottles until
 processing. Surface seawater (&lt; 5 m) samples were collected from
 Lat: 43.1822; Lon: -62.0983 (Station HL05) on May
 26<sup>th</sup>, 2022, and Lat: 41.4100; Lon: -60.6772
 (Station HL12) on May 23<sup>rd</sup>, 2022, in the Northwest
 Atlantic. Photodegradation experiments  
 Thirty-minute experiment: we added 8 pM of Me- and Ado-, and 5 pM
 of CN-B<sub>12</sub> and OH-B<sub>12</sub> into 15
 mL of seawater, collected at HL05, individually in UV-penetrable quartz
 vials (QP059, Cuvet.Co). Duplicate samples (15 mL) were taken directly
 after the addition of cobalamins at time 0. Then, duplicate quartz vials
 were exposed to direct, natural sunlight then 15 mL sample was transferred
 into clean amber vials in the dark at -20 °C after 5, 10, 15, and 30
 minutes on June 7<sup>th</sup>, 2022 (Ado-, Me- and
 CN-B<sub>12</sub>) (35.1°C; 1450
 µmol/s/m<sup>2</sup>) and July 21<sup>st</sup>,
 2022 (OH-B<sub>12</sub>) (26.6 °C; 1400
 µmol/s/m<sup>2</sup>). Four-day experiment: we added 4 pM
 OH-B<sub>12</sub> in 20 mL seawater, collected at HL12, in
 quartz vials, took duplicate T<sub>0</sub> samples then
 exposed to direct sunlight in May 2022. We transferred duplicate vials
 daily at noon into clean amber vials then stored in the dark at -20 °C
 until processing. Temperature (Logger, RC-5, Elitech) and light (Digital
 Light Meter, LUX29TK, TekcoPlus) were monitored (Table S2). For the 4-day
 experiment, mean day length was 11 hours, mean air temperature was 23.1 °C
 and average daytime light ~950 µmol/s/m<sup>2 </sup>with
 maximum 1400 µmol/s/m<sup>2</sup>. We collected dark controls
 that were supplemented with the same concentrations of cobalamin forms,
 wrapped in opaque black bags, exposed to similar temperatures, and sampled
 identically. We also processed control seawater samples in an identical
 manner to determine endogenous concentrations of cobalamins (Table
 S2). We performed all extractions in a dark, windowless
 room with only a red headlamp as a light source. Samples were extracted on
 100 mg HyperSep C18 solid phase extraction (SPE) cartridges
 (ThermoScientific, 60108-302) using a vacuum manifold. SPE cartridges were
 preconditioned with 2 × 0.5 mL MeOH then 2 × 0.5 mL deionized water and
 kept wet through the entire protocol. Samples were extracted at ~1 mL
 min<sup>-1</sup>, washed with 2 × 0.5 mL milli-Q water then
 eluted with 2 × 0.85 mL MeOH into clean microtubes. Eluent was dried for
 ~2 hours under vacuum (Vacufuge, Eppendorf, Ontario), in the dark, then
 stored at –80 °C prior to analysis when it was resuspended with 100 µL of
 H<sub>2</sub>O containing 0.1% formic acid.
 Degradation product identification experiment
 To investigate OH-B<sub>12</sub> degradation
 products, we prepared 1 µM of OH-B<sub>12</sub> in HPLC grade
 water and exposed it to ambient sunlight and temperature for 8 (EXP 1) and
 4 (EXP 2) days. One sample from EXP 2 on day 4 was exposed to two 5-minute
 intervals of 254 nm UVc light from a 30-Watt lamp (EXP 2, Day 4+UV). We
 diluted samples 200-fold before LC-MS analysis as outlined below and in
 supplemental methods. Mass spectrometry
 Cobalamin photodegradation in seawater was quantified using a
 Dionex Ultimate-3000 LC system coupled to an electrospray ionization
 source of a TSQ Quantiva triple-stage quadrupole mass spectrometer
 (ThermoFisher) with transition list reported in Table S4. Photodegradation
 products were investigated on an Agilent 1290 Infinity II LC coupled to a
 Q Exactive HF Orbitrap mass spectrometer (ThermoFisher) with a heated
 electrospray ionization probe using both data dependent acquisition and
 full scan modes. Complete details of LC-MS analyses are provided in
 supplemental methods. Cobalamin
 quantification We combined equal portions of each
 sample to obtain quality control (QC) pools for the 30-min and 4-day
 experiments. Cobalamins were quantified using standard addition with
 calibration curves prepared in the QC pools using the LC-MS approach
 described in the supplemental methods. Duplicate injections were performed
 with 0, 2.5, 5, and 25 fmol on C18 column for all cobalamin forms (Fig S7)
 and R<sup>2</sup> for slopes were all &gt;97%. SPE percent
 recovery for 100 mg HyperSep C18 cartridge (n = 2) was determined by
 spiking 15 mL seawater samples with each cobalamin form equivalent to 80
 fmol on C18 column (106 pM) before or after preconcentration on SPE. All
 reported concentrations and limits of detection are corrected for percent
 recoveries. Raw files generated with Xcalibur software (ThermoFisher) were
 uploaded into Skyline Daily (University of Washington) and the transitions
 with the best signal to noise and lowest interference were selected for
 quantification (Table S4). Limits of blanks and detection were determined
 according to Armbruster and Pry, 2008 (Table
 S3)<sup>62</sup>. Photodegradation product
 identification We identified potential
 photodegradation products of OH-B<sub>12</sub> in pure water
 using three approaches. First, we selected peaks of interest based on
 their increasing peak area in samples during sunlight exposure by
 performing a ‘background subtract’ of the first samples
 (T<sub>0</sub>) from T<sub>4</sub> and
 T<sub>8</sub> samples. Second, we assembled reports of
 cobalamin degradation products from the literature and searched for their
 accurate masses (±5 ppm) (Table S5). Third, we searched product ion
 spectra for masses that are known cobalamin fragments
 (<em>m</em>/<em>z</em> 147.0922
 and <em>m</em>/<em>z</em> 359.1005 product ions,
 DMB and DMB sugar-phosphate respectively). All candidate products were
 absent in blank samples.   We used exact mass to
 determine chemical formula of candidate degradation products by
 constraining upper limits with ring-double bond equivalent (&lt;26),
 elemental composition
 (C<sub>62</sub>H<sub>90</sub>CoN<sub>13</sub>O<sub>15</sub>P), and MS run-specific mass error (~4.0 ppm) of the OH-B<sub>12</sub> standard. We present level 2 (diagnostic) probable chemical structures (Fig 3) if they were either previously reported in literature or we were able to obtain a conclusive product ion spectrum <sup>45</sup>. Statistical Analysis Two outliers (Ado-B<sub>12</sub>: 15 min, CN-B<sub>12</sub>: 30 min) were removed from the photodegradation experiment analyses during data analysis due to abnormally high B<sub>12</sub> concentrations (Grubbs test, p-value &lt;0.05), likely due to contamination during extraction (Fig S1,). Differences between T<sub>0</sub> and dark controls at 30 min and 4 days (Table S4) as well as OH-B<sub>12</sub> at T<sub>0</sub> and T<sub>30</sub> (Fig 2D) were normally distributed and determined insignificant using a student t-test (Table S2).