Below detailed description was retreived from Publication: Fern�ndez-Acero FJ, Amil-Ruiz F, Dur�n-Pe�a MJ, Carrasco R, Fajardo C, Guarnizo P, Fuentes-Almagro C, Vallejo RA, Valorisation of the microalgae Nannochloropsis gaditana biomass by proteomic approach in the context of circular economy. J Proteomics, 193():239-242(2019) [pubmed]
MATERIALS AND METHODS
N. gaditana culture.
N. gaditana was kindly provided by Endesa Generacion S.A from its production facility in Carboneras, Almería, Spain. The algae were transferred to flasks containing f/2 media  at 21 - 28 ºC and 12 h light/12 h darkness using natural sea water (salinity 28 -30%) with a pH of 7.5 – 8, and collected when the biomass reached 0,8-0,9 gr/L.
In this study, two presentations of N. gaditana were used: fresh culture and atomized samples  as the common commercial presentation. Atomized sample was dried with a continuous hot air flow by a “Spray Dryer” (SPD-01) (fluid pressure: 4 bar; input temperature: 190 ºC; output temperature: 90 ºC; flow: 15 L/h).
Nannochloropsis spp. have a strong cellulose wall , that make difficult the extraction of proteins. For this reason, samples were subjected to an intense homogenization treatment prior to the protein extraction procedure. Optimal conditions were found by using mechanical lysis with Precellys® CK14, 7mL and a Minilys® homogenizer using three cycles of 30 seconds each one to 5000 rpm.
The protein precipitation was carried out using a previously described phenol-based procedure . All the assays were performed in triplicate. Once the protein extracts from N. gaditana were obtained, the protein concentration was estimated by fluorometric measurement using Qubit System ® (ThermoFisher, Inc.).
Protein extracts were cleaned-up in 1D SDS-PAGE at 10% polyacrilamyde. Previously, samples were concentrated by ultrafiltration using Amicon Ultra-0.5 Centrifugal Filter spin column with cut-off of 3kDa. Then, samples were loaded onto the stacking gel and 100 V was applied until the electrophoresis front reached the resolving gel. The run was stopped when the protein extract had entered 1 cm into the resolving gel and the gel was stained with Coomassie Blue. Protein bands were cut off, diced, and kept in water until digestion.
Briefly, gel dices were distained in 200 mM ammonium bicarbonate (AB)/50% acetonitrile for 15 min followed by 5 min in 100 % Acetonitrile. Protein was reduced by addition of 20 mM dithiothreitol in 25 mM AB and incubated for 20 min at 55 °C. The mixture was cooled down to room temperature, followed by alkylation of free thiols by addition of 40 mM iodoacetamide in 25 mM AB, in the dark for 20 min and the gel pieces were then washed twice in 25 mM AB. Proteolytic digestion was performed by addition of Trypsin (Promega, Madison, WI) at 12.5 ng/µl of enzyme in 25 mM AB and incubated at 37 ºC overnight. Protein digestion was stopped by addition of trifluoracetic acid at 1% final concentration and the digested samples were finally Speed vac dried.
Nano-LC was performed in a Dionex Ultimate 3000 nano UPLC (Thermo Scientific) with a C18 75 μm x 50 cm Acclaim Pepmap column (Thermo Scientific). The peptide mix was previously loaded on a 300um x 5 mm Acclaim Pepmap precolumn (Thermo Scientific®) in 2% acetonitrile/0.05% TFA for 5 min at 5µl/min. Peptide separation was performed at 40°C for all runs. Mobile phase buffer A was composed of water, 0.1% formic acid. Mobile phase B was composed of 80% acetonitrile, 0.1% formic acid. Samples were separated at 300 nl/min. Elution conditions were: 4-35%B for 60 min; 35-55% B for 6 min; 55-90% B for 3 min followed by 8 min wash at 90% B and a 15 minutes re-equilibration at 4%B. Total time of chromatography was 150 min.
Eluting peptide cations were converted to gas-phase ions by nano electrospray ionization and analysed on a Thermo Orbitrap Fusion (Q-OT-qIT, Thermo Scientific) mass spectrometer operated in positive mode. Survey scans of peptide precursors from 400 to 1500 m/z were performed at 120K resolution (at 200 m/z) with a 4 × 105 ion count target. Tandem MS was performed by isolation at 1.2 Da with the quadrupole, CID fragmentation with normalized collision energy of 35, and rapid scan MS analysis in the ion trap. The AGC ion count target was set to 2 x 103 and the max injection time was 300 ms. Only those precursors with charge state 2–5 were sampled for MS2. The dynamic exclusion duration was set to 15 s with a 10 ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The instrument was run in top 30 mode with 3s cycles, meaning the instrument would continuously perform MS2 events until a maximum of top 30 non-excluded precursors or 3s, whichever is shorter.
The raw data were processed using MaxQuant software (version 220.127.116.11) . MS2 spectra were searched with Andromeda engine against a database of Uniprot_Nannocloropsis_August2016 (“UniProt”). Peptides were generated by theoretical tryptic digestion allowing up to one missed cleavage, carbamidomethylation of cysteines as fixed modification and oxidation of methionine as variable modification. Precursor mass tolerance was 10 ppm and product ions were searched at 0.1 Da tolerance. In order to validate Peptide spectral matches (PSM) in Maxquant a target-decoy search strategy was applied, which integrates multiple peptide parameters such as length, charge, number of modifications and the identification score into a single quality that acts as the statistical evidence on the quality of each single peptide spectrum match. Protein quantification were carried out with MaxLFQ label-free quantification method . In the MaxLFQ label-free quantification method a retention time alignment and identification transfer protocol (“match-between-runs” feature in MaxQuant) is applied.
Differentially expressed proteins analysis was performed with the freely available software Perseus (version 18.104.22.168) . The peak intensities across the whole set of quantitative data for all the peptides in the samples were imported from the LFQ intensities of proteins from the MaxQuant analysis. Putative contaminant, decoy identifications and proteins only identified by site were filtered out from the imported raw data. Protein intensity quantifications were transformed to logarithmic scale in base two. Only those proteins quantified with high confidence (q-value < 0.01) in all analyzed samples were retained for further statistical analysis. Populations mean comparison by two-sample t-test, with permutation-based FDR, was employed to calculate statistical significance (threshold FDR < 0.05). Those proteins that showed a fold-change of at least 1.5 and satisfied an FDR < 0.05, were considered as differentially expressed. To identify proteins exclusively present in one population, those proteins quantified with high confidence (q-value < 0.01) in all replicates of a given sample, and not quantified in any replicate of the other sample, were considered as “exclusives” of the first sample. Differentially expressed proteins, overrepresented in one sample, together with the exclusive proteins quantified in such sample, were considered as proteins more abundant in that sample. In order to functionally describe the analysed samples, Blast2GO software was employed to evaluate enrichment in GO terms associated to each group of proteins.
RNA extraction, synthesis of cDNA, and PCR amplification.
Total RNA was isolated following the indications of the commercial kit NucleoSpin RNA® (Macherey-Nagel). To perform the procedure of synthesis of cDNA by RT-PCR, the indications of the commercial kit qScript® (Quanta Biosciences) were followed. For the PCR amplification procedure, the indications of the Phusion Flash High-Fidelity PCR Master Mix commercial kit (Thermo Scientific) were followed. The reaction conditions were as follows: 2x Phusion Flash PCR Master Mix 25 μL, 10 μM Prohi-F: 5'-GGC ATA TGT CTC CAG CAG GAC CGC TGG-3' 2.5 μL, 10 μM Prohi-R: 5'-GGG TCG ACC TAC CGC TTC TTT CCA GAC TTC-3' 2.5 μL, cDNA (65 ng/μL) 2 μL, and water 18 μL, for a total reaction volume of 50 μL. The same reaction conditions and the following pair of primers were applied for the amplification of the gene coding for the resistance to -phytophtora protein: Phyto-F: 5'-GG GCT AGC ATG TCC TTT TTG GTC AAT ACG C-3', Phyto-R: 5'-GGG TCG AC TCA CTT TCG CAC GCC CCG CT-3'. Both pairs of primers were designed with the OligoCalc program  with the Prohi-F and Prohi-R primers (GenBank: CM002468.1) and with Phyto-F and Phyto-R (GenBank: AZIL01000835.1), respectively. The thermocycler program was the following: initial denaturation at 98 oC for 10 sec, followed by 30 cycles at 98 oC for 1 sec, 72 oC for 5 sec, and 72 oC for 15 sec. A final extension cycle at 72 oC for 3 min and stop at 4 oC. The amplified products were visualized in an agarose gel (1%) run for 1 h at 110 V and stained with Gel-Red. Obtained PCR fragments were subsequently submitted to sequencing analysis (Stab VidaÓ, https://www.stabvida.com).