Dataset: CO2-stressed diatom proteome
Deployment: lab_Kustka

Culture conditions and photo-physiology upon CO2 shift
Cells were grown in media prepared from synthetic ocean water according to the Aquil recipe (Sunda et al. 2005) and maintained at 18 degrees C and 200 mmol photon m-2 s-1. Macronutrients were added at 100 mmol per liter NO3-,100 mmol per liter Si(OH)4 and 10 mmol per liter PO4-3. All medium preparation and sample handling was carried out in a class 100 laminar flow hood. All plastic ware was acid-cleaned by soaking in 10% trace metal grade HCl for 5 to10 days and rinsed with 18.2 M-ohm-cm deionized water. Media and culture flasks were microwave sterilized according to Keller et al. (1988) in polycarbonate bottles. Cell density was determined using a Coulter counter (Beckman-Coulter, Fullerton, CA, USA), and growth rates were computed from linear regressions of ln (cell density) versus time. Cells were acclimated to a constant CO2 (1740 mL per liter) and then shifted to 170 mL per liter CO2 within ~ 15 seconds. The pre-shift conditions were designed to provide a saturating supply of CO2 (1620 mL per liter or 58.6 mM compared to a diatom RuBisCO Km of roughly 30 mM; Badger et al. 1998) and obviate the need for CCM expression. This was done by maintaining cultures in house-built pH stats set at pH 7.61 (NBS scale) for >10 generations. At the shift, 1 N NaOH was added while mixing the culture bottle to achieve a pH of 8.48.  CO2 was calculated at these pH values using CO2SYS assuming a total alkalinity of 2.67 mM.  The pH stats were assembled as follows. A gel-filled combination pH electrode (Thermo, 9106BNWP) was mounted through the wall of a 1L polycarbonate bottle using a bulkhead mount. The electrode potential was monitored using a pH relay (Eutech Inst., pH200 controller). When the pH increased above a set threshold, the relay switched on a peristaltic pump (Master Flex C/L) and an aquarium air pump to deliver weak acid (0.03 N trace metal grade HCl) while mixing the culture.

Quantitative CO2 responsive proteomics
Cultures were grown under steady state conditions of low and high CO2 (~170 and 1740 mL per liter) in pH stats with Aquil media, as described above except that either low or high CO2 cells were grown with 15N- nitrate (>98%) or nitrate with a natural isotopic composition (i.e., 99.6% 14N). Two sets of independent biological replicates from each condition were processed. For each condition, ~900 mL were harvested at 4 -5 x 105 cells per mL and flash frozen in liquid nitrogen. Protein samples were extracted in 4% SDS, 7.5% glycerol, in 0.1 M NaCO3 and protease inhibitor (Sigma-Aldrich P-2714) and quantified before adding 0.1 mM DTT. Fifty micrograms of protein from each condition were mixed yielding a 1:1 ratio of 15N to 14N labeled protein mixture. Subsequent processing was carried out at the Biological Mass Spectrometry Facility of the UMDNJ-Robert Wood Johnson Medical School. The protein mixture was digested by trypsin.  Digested peptides were solubilized in buffer A (20mM ammonium formate, pH10) and subjected to high-pH Reverse phase HPLC (Gilson 306 pumps, 805 manometric module and uv/VIS 155 detector) equipped with a XbridgeTM C18 column (3.5um, 2.1x150mm, Waters, MA). The gradient used for separation of the peptides were 2% buffer B (20mM ammonium formate, 90% acetronitrile, pH10) for 2 min, then 2-45% in 43 min, 45 to 100% B in 5 min. 1 min fractions were collected and vacuum dried before combined or individually analyzed by nano-LC-MSMS.

NanoLC-MS/MS was performed using a RSLC system interfaced with a LTQ Orbitrap Velos (ThermoFisher, CA).  Samples were loaded onto a self-packed 100µm x 2cm trap packed with Magic C18AQ, 5µm 200 A (Michrom Bioresources Inc, CA) and washed with Buffer A( 0.2% formic acid) for 5 min with flowrate of 10ul/min. The trap was brought in-line with the homemade analytical column (Magic C18AQ, 3µm 200 A, 75 µm x 50cm) and peptides fractionated at 300 nL/min with a multi-stepped gradient (4 to 15% Buffer B (0.16% formic acid 80% acetonitrile) in 25 min and 15-25%B in 65 min and 25-50%B in 55 min). The mass spectrometer acquisition cycled through one MS in Orbitrap (resolution 60,000) followed by 20 MSMS (CID) in LTQ with dynamic exclusion (2 repeat count within 30 sec and exclusion time 60sec). The LC-MSMS data were analyzed using Proteome Discoverer software v1.3 (ThermoFisher). The data was first searched against T. pseudonana composed of sequences queried from Uniprot) using Sequest search engine through a "light" search assuming normal nitrogen isotope distribution and a "heavy" search assuming all amino acids were labeled with 15N. For both "heavy" and "light" searches, carbamidoethyl on cysteine was used as a fixed modification. For "light" search, oxidation of methionine was included as a variable modification. For "heavy" search, flexible modification of N-terminal modification of -0.997 Da was included as a variable modification. The identified peptides were quantified with custom built precursor ion quantification method within the software.