Microalgae that were cultivated in outdoor pond (15 m long x 5 m width x 40 cm depth) which were used as fish feeds in the area of Pengjing city, Liaoning province, China were collected and purified through serial dilution. One of the purified isolates, namely PJ12, was analyzed in this study. The clonal strain was cultivated in modified f/2 medium (in 1 L, it contains 30 g sea salt, 150 mg NaNO3, 10 mg NaH2PO4·H2O, 3.15 mg FeCl3·6H2O, 4.16 mg Na2EDTA·2H2O, 10 μg CuSO4·5H2O, 6 μg Na2MoO4·2H2O, 22 μg ZnSO4·7H2O, 10 μg CoCl2·6H2O, and 180 μg MnCl2·4H2O, 2.5 μg Vitamin B12, 2.5 μg biotin, and 0.5 μg thiamine HCl)37 (link) in glass flask with orbital shaking at 100 rpm, 25 °C, under continuous illumination of 50 µM photon m−2 s−1. Cell density was determined gravimetrically using a weighing balance (AG204, Mettler-Toledo Inc., Columbus, OH, USA). after collecting 50–100 ml cells on filter paper and oven-dry overnight. Cells at log-phase were subjected to medium shift to f/2 without nitrate and with nitrate. Cells three days after medium shift were collected for RNA-seq analysis with a PE100 protocol on the BGISEQ-500 machine in BGI (Beijing Genomics Institute, Shenzhen, China) and for Lipidomic analysis with a Thermo Orbitrap Elite mass spectrometer (Thermo Fisher, Waltham, MA, USA) in SSB (Shanghai Sensichip Biotech Co. Ltd., Shanghai, China).
Thermo orbitrap elite
Manufactured by Thermo Fisher Scientific
Sourced in China, Germany
The Thermo Orbitrap Elite is a high-resolution mass spectrometer designed for advanced analytical applications. It utilizes Orbitrap technology to provide accurate mass measurements and high-resolution capabilities for the detection and identification of a wide range of molecular species.
Automatically generated - may contain errors
Lab products found in correlation
Ag204, by Mettler Toledo (1 mentions)
Pack polyamine 2, by YMC (1 mentions)
Acquity uplc 1 class system, by Waters Corporation (1 mentions)
Acquity uplc protein beh c4 column, by Waters Corporation (1 mentions)
Protein deconvolution 2, by Thermo Fisher Scientific (1 mentions)
Orbitrap elite mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Rapiflex, by Bruker (1 mentions)
Instantblue, by Abcam (1 mentions)
8 protocols using thermo orbitrap elite
1
Microalgae Cultivation and Transcriptomic Analysis
2
Quantitative Proteomics of Caenorhabditis elegans
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Heavy- and light-labeled samples of the genotypes—wild type, mir-58.1, mir-80; mir-58.1, and mir-80; mir-58.1; mir-81-82—were prepared by feeding with heavy- and light-labeled bacteria, respectively. Worms were harvested at the late L4 stage for subsequent protein and RNA isolation. Three biological replicates were grown, and the experiment was performed in two variants: one without fractionation (in three biological replicates) and the other with HILIC fractionation (one replicate). Proteins from all the samples were extracted with 50 mM Tris/HCl (pH 8.3), 5 mM EDTA, 8 M urea buffer, and glass beads as described previously (Schrimpf et al. 2009 (link)). Peptides obtained from SILAC samples were fractionated with a Agilent 1100 HPLC flow system using a 4-mm HILIC column YMC-pack polyamine II (YMC Europe GmbH). Samples were analyzed by LC-MS/MS using a Thermo easy nLC 1000 HPLC system coupled to a Thermo Orbitrap elite hybrid mass spectrometer equipped with a Nanoflex electrospray source (Thermo Fisher Scientific). Further details on SILAC sample preparation, protein isolation, HILIC fractionation, and mass spectrometric measurements can be found in the Supplemental Methods.
3
Intact Protein Mass Analysis by UPLC-MS
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The intact masses of the proteins were analyzed using a Waters ACQUITY I class UPLC system (Milford, MA) with an ACQUITY UPLC Protein BEH C4 column (2.1 mm × 100 mm, 1.7 μm particle size; Waters). The mobile phases were 0.1% formic acid in water (eluent A) and 0.1% formic acid in acetonitrile (eluent B). The gradient applied was: 0–3 min, 5% eluent B; 3–13 min, linear increase to 50% eluent B at 0.2 ml/min. The eluent was injected into a Thermo Orbitrap Elite (Thermo Fisher Scientific, Waltham, MA) and ionized with an electrospray source. MS spectra were acquired in the mass range of 400–2,000 m/z and 120,000 resolution at m/z 200. The deconvoluted mass spectra were generated using Protein Deconvolution 2.0 (Thermo Fisher Scientific, Waltham, MA).
4
Mass Spectrometry-based Hydrogen-Deuterium Exchange
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Mass spectra were measured by a Thermo
Orbitrap Elite (Thermo Fisher Scientific; Waltham, MA). The instrument
settings were spray voltage, 3.7 kV; sheath gas flow rate, 25 (arbitrary
units); capillary temperature, 275 °C. In the Orbitrap stage,
MS spectra were acquired with the resolution set at 60000, which has
been shown to yield accurate measurements of hydrogen and deuterium
composition.65 (link) Extracted ion chromatograms
(XICs) of undeuterated samples were used for automated peak area calculation
in Xcalibur. From mass spectra obtained during HDX-MS experiments,
the centroid of each deuterated peptide envelope and the relative
deuterium uptake by each peptide was calculated by HDX WorkBench.66 (link)
Orbitrap Elite (Thermo Fisher Scientific; Waltham, MA). The instrument
settings were spray voltage, 3.7 kV; sheath gas flow rate, 25 (arbitrary
units); capillary temperature, 275 °C. In the Orbitrap stage,
MS spectra were acquired with the resolution set at 60000, which has
been shown to yield accurate measurements of hydrogen and deuterium
composition.65 (link) Extracted ion chromatograms
(XICs) of undeuterated samples were used for automated peak area calculation
in Xcalibur. From mass spectra obtained during HDX-MS experiments,
the centroid of each deuterated peptide envelope and the relative
deuterium uptake by each peptide was calculated by HDX WorkBench.66 (link)
5
Bacterial Metabolite Extraction and Analysis
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Bacteria were grown until OD600 = 0.5−0.8, following which the number of bacteria was adjusted to 108 CFU, and extracted using extract solution (methanol: acetonitrile: ddH2O = 2:2:1 with 2 mg/L 2-chloro-l -phenylalanine), then homogenized using 5 μm glass beads. The sample was centrifuged and vacuum dried then reconstituted with 50% acetonitrile and storage at − 80 °C until for analysis. Each sample (10 μL) was injected into a vanquish-focused ultra-high-performance liquid chromatography (UHPLC) system (Thermo Orbitrap Elite) coupled with an Orbitrap Elite Mass Spectrometer (Thermo Fisher Scientific) using electrospray ionization. Please see Additional file 1 for more detail methods.
6
Lipid MALDI-MSI for Comprehensive Identification
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Lipid MALDI-MSI was performed in negative ionization mode on a Bruker RapifleX (Bruker Daltonik, GmbH, Germany) and on a Thermo Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with an elevated-pressure MALDI ion source (Spectroglyph LLC, Kennewick, WA, USA), for subsequent lipid identification. In short, Bruker RapifleX MSI experiments were performed at 50 µm spatial resolution in reflector mode with 500 shots per pixel. Lipids were analyzed in the 200−1800 m/z range using red phosphorus as an internal calibrant.
Subsequent tandem MS for lipid identification was performed as described by Ellis et al., 2018 (44 (link)). To elaborate, MSI experiments were conducted at 25 µm (horizontal) and 50 µm (vertical) spatial resolution step-sizes. Subsequently, parallel full-scan FTMS (Orbitrap) and IT-MS/MS (ion trap) scans at adjacent 25-µm positions were conducted at a mass resolution of 240,000 and lipids were analyzed in the 200 – 1600 m/z range.
Subsequent tandem MS for lipid identification was performed as described by Ellis et al., 2018 (44 (link)). To elaborate, MSI experiments were conducted at 25 µm (horizontal) and 50 µm (vertical) spatial resolution step-sizes. Subsequently, parallel full-scan FTMS (Orbitrap) and IT-MS/MS (ion trap) scans at adjacent 25-µm positions were conducted at a mass resolution of 240,000 and lipids were analyzed in the 200 – 1600 m/z range.
7
Assessing Deuterium Uptake by HDX-MS
8
Biotinylated Protein Identification by LC-MS/MS
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Affinity-purified biotinylated protein samples were briefly separated by SDS PAGE, then fixed and stained using InstantBlue (Expedeon). Gel fragments containing protein samples were digested with trypsin and peptides extracted under standard conditions. Peptides were subjected to liquid chromatography tandem mass spectrometry (LC–MS/MS) using a Thermo Orbitrap Elite coupled with a Thermo nanoRSLC system (Thermo Fisher Scientific). Peptides were selected for fragmentation automatically by data-dependent analysis. Raw data were processed using Progenesis QI (v4.1, Nonlinear Dynamics) and searched against the SwissProt and Trembl databases (accessed July, 2017). Database searches were performed against the human, mouse and E. coli proteome databases with tryptic digest specificity, allowing a maximum of two missed cleavages, and an initial mass tolerance of 5 ppm (parts per million). Carbamidomethylation of cysteine was applied as a fixed modification, while N-acylation and oxidation of methionine, as well as the biotinylation of lysine, were applied as variable modifications during the database searches.
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