Below detailed description was retreived from Reference: Wei L, El Hajjami M, Shen C, You W, Lu Y, Li J, Jing X, Hu Q, Zhou W, Poetsch A, Xu J. Transcriptomic and proteomic responses to very low CO2 suggest multiple carbon concentrating mechanisms in Nannochloropsis oceanica. Biotechnol Biofuels. 2019 12:168, PubMed: 31297156.
Culture conditions of N. oceanica
Nannochloropsis oceanica IMET1 was inoculated into the modified f/2 liquid medium, which was prepared with 35 g L−1 sea salt (Real Ocean, USA), 1 g L−1 NaNO3, 67 mg L−1 NaH2PO4*H2O, 3.65 mg L−1 FeCl3*6H2O, 4.37 mg L−1 Na2EDTA*2H2O, trace metal mix (0.0196 mg L−1 CuSO4*5H2O, 0.0126 mg L−1 NaMoO4*2H2O, 0.044 mg L−1 ZnSO4*7H2O, 0.01 mg L−1 CoCl2 and 0.36 mg L−1 MnCl2*4H2O) and vitamin mix (2.5 µg L−1 VB12, 2.5 µg L−1 biotin and 0.5 µg L−1 thiamine HCl) [52]. The cells were first cultured in f/2 medium at 25 °C with 80 ± 5 μmol m−2 s−1 continuous irradiation in a 1-L column reactor (inner diameter 5 cm). The seed cultures were bubbled with 5% CO2. At the logarithmic phase (OD750 = 3.0), cells were harvested by centrifugation and then washed with fresh medium, before being used for the following experiments.
In total, six identical column reactors were employed for the wild-type N. oceanica culture. Each reactor contains 800 mL of fresh modified f/2 liquid medium, which was supplemented with 10 mM Tris–HCl buffer (pH = 8.2) in order to accurately control the pH during the culture. Equal numbers of the seed cells from six independent reactors were re-inoculated into each of the six new column reactors with fresh medium to an OD750 of 1.5, respectively. The light intensity was maintained at 80 ± 5 μmol m−2 s−1. The six algal cultures were first aerated with air enriched with 5% CO2 (“high-CO2” conditions, or HC) for 1 h. After the preadaption phase, three of the algal cultures proceeded under HC as the control condition, whereas the other three were switched to aeration with 0.01% CO2 (“very low-CO2” conditions, or VLC; the customized CO2 gas was provided by Dehai Gas Company, China) for CCM induction (Additional file 1: Figure S1; [17, 48]). After switching to the designated culture condition (e.g., VLC), cell aliquots were taken at 0, 3, 6, 12 and 24 h from each column by syringe for physiological measurement (including OD, inorganic carbon concentration, chlorophyll content, photosynthetic rate, etc.), transcriptomic profiling, proteomic profiling and metabolite analysis. Three biological replicates of algal cultures, corresponding to the collectively six column reactors, were established under each of the above VLC and HC conditions, respectively.
Proteome sampling, sequencing and analysis
Nannochloropsis oceanica cells were collected by centrifugation at 2500 g at 4 °C. Total proteins were extracted with plant protein extraction kit (CWBIO, Beijing) and quantified by the approach of BCA protein assay (CWBIO, Beijing). Protein samples were loaded onto 12.5% (v/v) polyacrylamide gels containing 0.4% (w/v) SDS (50–100 µg sample per lane). The gels were run at room temperature, 300 V and 30 mA until all proteins migrated about 1 cm into the separation gel Proteins were visualized with a coomassie brilliant blue (CBB-G250) stain as previously described [65]. Protein bands were excised from the gels, cut into small cubes (ca. 1 × 1 mm3) and destained [66]. Gel pieces were dried in a SpeedVac and immersed completely in digestion solution (~ 200 µL). The digestion solution consisted of sequencing grade modified trypsin (Promega, USA), which was diluted in 40 mM ammonium bicarbonate (pH 8.6) to a concentration of 12.5 ng µL−1.
The protein digestion was performed overnight at 37 °C with tempered shaker (HLC MHR20, 550 rpm). After digestion, the samples were centrifuged and supernatants were transferred to LC–MS grade glass vials (12 × 32 mm2 glass screw-necked vial, Waters, USA). The extracted peptides were dried using a SpeedVac and stored at room temperature. Prior to MS analysis, peptides were re-suspended in 20 µL of buffer A (0.1% formic acid in water, ULC/MS; Biosolve, the Netherlands) by sonication for 10 min and used for MS analysis. Each measurement was taken with 8 μL of sample.
An UPLC HSS T3 column (1.8 mm, 75 mm, 150 mm, Waters, USA) and an UPLC Symmetry C18 trapping column (5 mm, 180 mm, 20 mm, Waters, USA) for LC as well as a PicoTip Emitter (SilicaTip, 10 mm i.d., New Objective, USA) were used in combination with the nanoACQUITY gradient UPLC pump system (Waters, USA) coupled to a LTQ Orbitrap Elite mass spectrometer (Thermo Scientific, USA). For elution of the peptides, a gradient with increasing concentration of buffer B (0.1% formic acid in acetonitrile, ULC/MS, Biosolve, the Netherlands) was used in 105 min at a flow rate of 400 nL/min and a spray voltage of 1.6 kV: 0–5 min: 2% buffer B; 5–10 min: 2–5% buffer B; 10–71 min: 5–30% buffer B; 72–77 min: 85% buffer B; 77–105 min: 2% buffer B. The analytical column oven was set to 55 °C, and the heated desolvation capillary was set to 275 °C. The LTQ Orbitrap Elite was operated via instrument method files of Xcalibur (Rev. 2.1.0) in positive ion mode. The linear ion trap and Orbitrap were operated in parallel, i.e., during a full MS scan on the Orbitrap in the range of 150–2000 m/z at a resolution of 60,000 MS/MS, spectra of the 20 most intense precursors were detected in the ion trap using the rapid scan mode. The relative collision energy for collision-induced dissociation (CID) was set to 35%. Dynamic exclusion was enabled with a repeat count of 1 and a 45 s exclusion duration window. Singly charged ions of unknown charge state were rejected from MS/MS.
Protein identification for VLC and HC data was performed with Proteome Discoverer using Sequest HT. Protein identification for wild-type data was performed by Andromeda search engine [67] embedded in MaxQuant [68, 69], which searched against the complete proteome database of N. oceanica IMET1 [20]. The mass tolerance for precursor ions was set to 10 ppm; the mass tolerance for fragment ions was set to 0.4 Da. Only tryptic peptides with up to two missed cleavages were accepted. The oxidation of methionine, acetylation on N-terminal and propionamide on cysteine was admitted as a variable peptide modification. The false discovery rate (FDR, q value) of protein identification was set to 1% and was determined with the percolator validation in Proteome Discoverer (for VLC and HC).
For the VLC versus HC comparison, un-normalized PSM values were imported into Perseus software. After a log2 transformation, the samples were normalized on the median value of each sample for comparability. Then a two-way ANOVA was employed as mentioned for wild-type data analysis. For each time point, a two-sample t test was conducted with a S0 value of 0.1 and an FDR of 0.05; a q value was also reported during this analysis. All the other analyses were carried out in the MATLAB® environment for statistical computing and graphics.
It has been well recognized that correlation between proteomic data and transcriptomic data is not necessarily high, i.e., 50% [70, 71]; moreover, protein expression is usually delayed in plants as compared to transcriptomic data [72]. Therefore, here in most cases, the more comprehensive transcriptome data were exploited for further analyses. However, important findings were supported by both transcriptomics and proteomics, e.g., the strongly increased expression of CA5 in biophysical CCM and PPDK2 in biochemical CCM.