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Methodology
\nThe following are excerpts from Sun et al., 2018. Please refer to this publication for more details. <\/p>\n
Non-axenic Scrippsiella trochoidea CCMP 3099 was originally obtained from the National Center for Marine Algae and Microbiota (Provasoil-Guillard NCMA, Boothbay Harbor, ME, United States). For maintenance, cultures were grown in L1 seawater media (Guillard and Ryther, 1962; Guillard et al., 1973; Guillard and Hargraves, 1993). Medium was prepared from natural seawater collected near Key West (salinity of 33), which was aged for at least 6 month in the dark and autoclaved. Maintenance and experimental cultures were grown in a light\/dark incubator at 23\u201324\u00b0C and 30\u201340 \u03bcmol quanta\u22c5m-2 s-1 light under a 12-h light:12-h dark cycle.<\/p>\n
For the light\/dark comparisons, cultures were grown under replete conditions in full L1 media containing 880 \u03bcM NaNO3 and 36 \u03bcM NaH2PO4 (N\/P ratio 24:1). Experimental cultures were started as a 1:10 inoculum from exponentially growing cultures into 1 L of media in 2.5 L Pyrex Fernbach flasks without shaking and monitored daily via cell counts and chlorophyll measurements. Cell counts were conducted by addition of 1% Lugol\u2019s iodine and direct counting of cells in 96-well microtiter plates using a dissection microscope. Dilutions were made as necessary and at least 10 wells containing 100 \u03bcL diluted culture were counted to average cell counts. Chlorophyll a was measured via fluorometry (Welschmeyer, 1994) by filtering 5 mL of culture in triplicate onto GF\/F filters, over-night extraction with methanol, and quantification using a Turner Trilogy Laboratory Fluorometer. Cultures were grown into late-log phase (data not shown) and then sampled 3 h before and 3 h after the light was turned on. Cultures were sub-sampled for MS analysis, making sure to keep \u2018dark\u2019 sample exposure to light to a minimum by wrapping sampling tubes in aluminum foil.<\/p>\n
N-deplete cultures were generated by first growing cells on L1 media with an N:P ratio of 2.4:1 (88 mM nitrate: 36 mM phosphate) analogous what has been previously described (Harke et al., 2017). This lower ratio stoichiometrically limited cultures in nitrogen and at least three transfers were performed to ensure no carryover from higher nutrient full L1 medium. Cultures were monitored daily via cell counts (see above), chlorophyll a quantification (see above), and quantification of nitrate\/nitrite via a Vanadium reduction method (Miranda et al., 2001). Parallel cultures were set up in which one culture was allowed to run out of nitrogenous nutrients (N-deplete), while the culture (control) was fed additional nitrate every second day to bring total nitrate\/nitrite concentrations back to starting levels. Once nitrate\/nitrite levels dropped below the limit of detection (\u223c1\u20132 \u03bcM) in the N-deplete culture, cultures were grown for an additional 24 h before sampling to ensure that N-depletion was complete. Both the N-deplete and replete cultures were then sampled for MS analysis of single cells.<\/p>\n
Individual cells of S. trochoidea were analyzed via the \u2018Single-probe\u2019 MS techniques.
\nFor the analysis, cells were deposited onto 0.2 \u03bcm polycarbonate membranes by gentle filtration, and the cells were rinsed with phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4; pH of 7.4) to remove culture medium. Filters were then placed on a home-built XYZ-translation stage system and spatial motion was controlled by a custom designed LabView software package (Lanekoff et al., 2012). The Single-probe tip (<10 \u03bcm) was then precisely insert into single S. trochoidea cells (typically \u223c20\u201330 \u03bcm cellular diameter) using a microscope as a guide. During the experiment, a syringe (250 \u03bcl; Hamilton, Co., Reno, NV, United States) was used to continuously provide the sampling solvent (acetonitrile; Sigma-Aldrich, St. Louis, MO, United States), and a liquid junction formed at the Single-probe tip to perform highly efficient extraction of cellular contents. The analytes were withdrawn by capillary action toward the nano-ESI emitter, and ionized for analysis using a Thermo LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Waltham, MA, United States). Mass analyze parameters were as follows: mass resolution 60,000, +4 kV ionization voltage at positive ion mode (0.05\u20130.07 \u03bcA of ion current), 1 microscan, 100 ms max injection time, and automatic gain control on.<\/p>\n
Data processing:
\nThe Thermo Xcalibur Qual Browser (Thermo Scientific, Waltham, MA, United States) was used to export MS data (m\/z values with relative intensities) as tab-delimited data files. As a conservative approach, only relatively abundant peaks with ion intensities > 103 were exported. This approach excluded 6% of low signal peaks as background while retaining 94% of total signal intensity. The relative ion intensities were normalized to the total ion current to minimize the influence induced by fluctuations of ion signals during experiments. The Geena 2 online software tool1 was then used for peak alignment (Romano et al., 2016), and the aligned m\/z values were then used for comparisons. Parameters used in Geena 2 include analysis range (from 100 to 1500 m\/z), maximum number of isotopic replicas (5), maximum delta between isotopic peaks (0.05 Da), and maximum delta for aligning replicates (0.01 Da). Metaboanalyst 3.02 was used to conduct statistical data analysis, including PLS-DA (partial least squares discriminant analysis) and t-tests (Xia et al., 2015; Xia and Wishart, 2016). PLS-DA was used to visualize differences in chemical composition profiles among treatment groups, while t-tests were applied to extract molecular peaks with significant abundance changes (p < 0.05). Finally, the online database METLIN3 was used to tentatively label all ions of interest (Smith et al., 2005; Guijas et al., 2018), to perform hierarchical clustering, and to generate heat maps. Lastly, Pathos4 was used to attempt identification of significantly regulated metabolic pathways by considering all KEGG maps in all organisms (Leader et al., 2011).<\/p>\n
Single-Probe description:
\nDetailed fabrication protocols of the Single-probe have been previously described (Pan et al., 2014; Rao et al., 2015; Sun et al., 2017). Briefly, a Single-probe has three components: a dual-bore quartz tubing [outer diameter (OD) 500 \u03bcm; inner diameter (ID) 127 \u03bcm, Friedrich & Dimmock, Inc., Millville, NJ, United States] pulled using a laser pipette puller (P-2000 micropipette puller, Sutter Instrument, Novato, CA, United States), a fused silica capillary (OD 105 \u03bcm; ID 40 \u03bcm, Polymicro Technologies, Phoenix, AZ, United States), and a nano-ESI emitter made from the same type of fused silica capillary. A Single-probe is fabricated by embedding a fused silica capillary and a nano-ESI emitter into both of the channels of the laser-pulled dual-bore quartz needle.<\/p><\/div>","@type":"rdf:HTML"}],"http:\/\/purl.org\/dc\/terms\/description":[{"@value":"
These data were published in Sun et al., 2018.<\/p>\n
A tabular version of this dataset is accessible\u00a0by clicking the \"Get Data\" button on this page.\u00a0 The columns of the tabular dataset are described in the \"Parameters\" section on this page.\u00a0 This dataset is available as\u00a0standard MS file format (.mzML) files within the following tar.gz file: .mzML is a standard MS file format that can be viewed using freeware such as\u00a0mMass\u00a0(http:\/\/www.mmass.org\/<\/a>) and ProteoWizard (http:\/\/proteowizard.sourceforge.net\/index.shtml<\/a>).<\/p><\/div>","@type":"rdf:HTML"}],"http:\/\/www.w3.org\/2000\/01\/rdf-schema#label":[{"@value":"Scrippsiella Metabolic Fingerprints","@type":"xsd:string"}],"http:\/\/ocean-data.org\/schema\/hasProcessingDescription":[{"@value":" BCO-DMO Data Manager Processing Notes:
Algal_Cell_Data_mzML.tar.gz<\/a>\u00a0(2.4 GB, contains 20 .mzML files)<\/p>\n
\n* added a conventional header with dataset name, PI name, version date
\n* modified parameter names to conform with BCO-DMO naming conventions<\/p><\/div>","@type":"rdf:HTML"}],"http:\/\/purl.org\/dc\/terms\/identifier":[{"@value":"746185","@type":"xsd:int"}],"http:\/\/purl.org\/dc\/terms\/title":[{"@value":"Scrippsiella Metabolic Fingerprints"}],"http:\/\/purl.org\/dc\/terms\/date":[{"@value":"2018-09-14T12:32:38-04:00","@type":"xsd:dateTime"}],"http:\/\/purl.org\/dc\/terms\/created":[{"@value":"2018-09-14T12:32:38-04:00","@type":"xsd:dateTime"}],"http:\/\/purl.org\/dc\/terms\/modified":[{"@value":"2023-07-07T16:10:26-04:00","@type":"xsd:dateTime"}],"http:\/\/rdfs.org\/ns\/void#inDataset":[{"@id":"http:\/\/www.bco-dmo.org\/"}],"http:\/\/ocean-data.org\/schema\/namedGraph":[{"@value":"urn:bcodmo:dataset:746185","@type":"xsd:token"}],"http:\/\/ocean-data.org\/schema\/osprey_page":[{"@id":"https:\/\/www.bco-dmo.org\/dataset\/746185"}],"http:\/\/ocean-data.org\/schema\/identifier":[{"@value":"_:Identifier746185"}],"http:\/\/ocean-data.org\/schema\/datasetTitle":[{"@value":"Metabolomic fingerprints of individual algal cells using the Single-Probe Mass Spectrometry technique from experiments conducted between May and August of 2017","@language":"en-US"}],"http:\/\/ocean-data.org\/schema\/abstract":[{"@value":"Single-probe Mass Spectrometry was used to obtain mass to charge ratio (m\/z) and relative intensity values of marine dinoflagellate Scrippsiella trochoidea cells grown under different illumination levels and under nitrogen (N) limiting conditions.","@language":"en-US"}],"http:\/\/purl.org\/dc\/terms\/rights":[{"@id":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"http:\/\/ocean-data.org\/schema\/deprecated":[{"@value":"false","@type":"xsd:boolean"}],"http:\/\/ocean-data.org\/schema\/temporalExtent":[{"@value":"_:temporalExtent746185"}],"http:\/\/purl.org\/dc\/terms\/bibliographicCitation":[{"@value":"Wawrik, B., Yang, Z. (2018) Metabolomic fingerprints of individual algal cells using the Single-Probe Mass Spectrometry technique from experiments conducted between May and August of 2017. Biological and Chemical Oceanography Data Management Office (BCO-DMO). 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