assays/2012_proteome_data_bmfz_stefanski/protocols/05_proteome_dataAnalysis.md

-# Computational MS data analysis
-
-For peptide and protein identification the acquired MS spectra were analyzed using the MaxQuant version 1.3.0.5 (MPI for Biochemistry, Planegg, Germany) with default parameters {Cox:2008ir}. Quantification was performed using the unlabeled quantification option of MaxQuant. The identified spectra were matched against the Ricinus proteome using the peptide search engine Andromeda {Cox:2011eg}. Only proteins containing at least two unique peptides and a minimum of three valid values in at least one group were quantified. A full list of all identified peptides from the proteome experiment is presented in supplemental Table S1. The raw files and identified spectra were submitted to ProteomeXChange (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository and can be accessed under the identifier PXDXXXXXX.
-
-All identified Ricinus proteins were analyzed by bidirectional BLAST against the Arabidopsis proteome {Altschul:1990vt}. Organelle distribution within the collected fractions was assayed using a set of marker proteins. Proteins were assigned as organelle markers if the experimental localization of their Arabidopsis homologues in the SUBA 4.0 database {Hooper:2017cf} corresponded with their sequence-based localization prediction in Ricinus. We predicted protein localization to peroxisomes, mitochondria, and plastids manually and using the publicly available tools PPero, PredPlantPTS1, and TargetP (Emanuelsson et al. 2000; Reumann et al., 2012; Wang et al. 2017).

assays/2_2-FAME-analysis/README.md

+
+## FAME-analysis
+
+![fpls-14-1182105-g002.jpg](dataset/fpls-14-1182105-g002.jpg)
+
+**Figure 2**
+
+Storage oil mobilization in castor bean endosperm. (A) Relative fresh weight of the endosperm in dry seeds (-1d), imbibed seeds (0d) and 1- to 7-day old dark-grown castor bean seedlings (1-7d) as percentage of fresh weight of the whole plant. Data represent arithmetic means ± SD of 3 biological replicates. (B) Relative ricinoleic acid content of the endosperm from seeds and seedlings. Samples were normalized to the average fresh weight of on endosperm and expressed as percentages of their initial quantities determined in dry seeds (-1d). Error bars show standard deviations of the means of at least three biological replicates.
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assays/2_3-OrganellesFromEndosperm/README.md

+
+
+![fpls-14-1182105-g003.jpg](dataset/fpls-14-1182105-g003.jpg)
+
+**Figure 3** 
+
+Isolation of organelles from etiolated castor bean seedlings. Four fractions of the sucrose density step gradient after centrifugation were taken from the gradient for various analyses at the interface 30% - 44% (w/w) sucrose solution (F1), 44% - 48% (w/w) sucrose solution (F2), 48% - 49% (w/w) sucrose solution (F3), and 50% - 54% (w/w) sucrose solution (F4).
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assays/2_4-EnzymeActivityOrganellarMarkers/README.md

+![fpls-14-1182105-t001.jpg](dataset\fpls-14-1182105-t001.jpg)
+
+**Table 1**
+
+Distribution of marker enzyme activities between the fractions.
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assays/2_4-EnzymeActivityOrganellarMarkers/protocols/EnzymeActivityOrganellarMarkers.md

+# 2.4 Measurements of enzyme activity of organellar marker enzymes
+
+The distribution of peroxisomes, mitochondria, and plastids within the four organellar fractions were examined using enzymatic marker proteins. All enzyme assays were performed photospectrometrically in a plate reader (SynergyH1, BioTek) at room temperature. For each sample three technical replicates were measured. Total protein concentration of the fractions was determined using the Pierce BCA protein assay kit (ThermoFisher Scientific). Enzyme activity was expressed as units per mg total protein. The activities of the following marker enzymes were analyzed: Catalase for peroxisomes (Breidenbach et al., 1968), fumarase for mitochondria (Nishimura and Beevers, 1981), and phosphoglycerate dehydrogenase for plastids (Benstein et al., 2013).
+
+- Breidenbach, R. W., Kahn, A., and Beevers, H. (1968). Characterization of glyoxysomes from castor bean endosperm. Plant Physiol. 43, 705–713. doi: 10.1104/ pp.43.5.705
+- Nishimura, M., and Beevers, H. (1981). Isoenzymes of sugar phosphate metabolism in endosperm of germinating castor beans. Plant Physiol. 67, 1255–1258. doi: 10.1104/ pp.67.6.1255
+- Benstein, R. M., Ludewig, K., Wulfert, S., Wittek, S., Gigolashvili, T., Frerigmann, H., et al. (2013). Arabidopsis phosphoglycerate dehydrogenase1 of the phosphoserine pathway is essential for development and required for ammonium assimilation and tryptophan biosynthesis. Plant Cell 25, 5011–5029. doi: 10.1105/tpc.113.118992
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assays/2_5-OrganellarMembraneIsolation/protocols/OrganellarMembraneIsolation.md

+# 2.5 Isolation of organellar membranes
+
+Membranes were isolated from the collected organellar fractions (F1-4) as described by Fujiki et al. (1982) and Reumann et al. (1995) with modifications. The obtained fractions were slowly diluted with hypo-osmotic buffer (20 mM HEPES-KOH, pH 6.8 and 0.8 mM MgCl2) and incubated on ice for 30 minutes to osmotically disrupt the organelles. The suspension was subjected to 10 freeze/thaw cycles (freezing in liquid nitrogen, thawing at room temperature) to lyse efficiently the organelles. After each thaw cycle the sample was homogenized by mixing. Membranes were sedimented by centrifugation at 100,000 g at 4°C for 1 h and washed in 100 mM sodium carbonate (pH 11.5) to remove membrane- associated proteins. A second centrifugation step (100,000 g at 4°C for 1 h) was performed to harvest the organellar membranes. The membrane pellet was resuspended in 20 mM HEPES-KOH, pH 6.8 and 0.8 mM MgCl2 and stored at -80°C for further experiments. Concentration of the membrane proteins were determined using Pierce BCA protein assay kit (ThermoFisher Scientific).
+
+- Fujiki, Y., Hubbard, A. L., Fowler, S., and Lazarow, P. B. (1982). Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J. Cell Biol. 93, 97–102. doi: 10.1083/jcb.93.1.97
+- Reumann, S., Maier, E., Benz, R., and Heldt, H. W. (1995). The membrane of leaf peroxisomes contains a porin-like channel. J. Biol. Chem. 270, 17559–17565. doi: 10.1074/jbc.270.29.17559
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