add protocols

assays/2012_proteome_data_bmfz_stefanski/protocols/01_plant_growth.md

+
+# Plant growth conditions
+
+Dry seeds of *Ricinus communis var. zanzibariensis* were surface-sterilized in a solution of 0.1% (w/v) 8-quinolinol in water for ten minutes and soaked in running tap water over night for seed imbibition. The imbibed seeds were placed on moist vermiculite and incubated at 30°C in the dark {Beevers:1974ug}.

assays/2012_proteome_data_bmfz_stefanski/protocols/03_membrane_isolation.md

+# 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 {Fujiki:1982gt} {Reumann:1995wh}. 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 vortexing. 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).

assays/2012_proteome_data_bmfz_stefanski/protocols/04_proteome_massSpec.md

+# Proteome acquisition via mass spectrometry analysis
+
+Total proteins and the enriched membrane proteins of the organellar fractions (F1-4) isolated from Ricinus endosperm tissue were analyzed by mass spectrometry (MS). Therefore, protein samples were loaded on an SDS-polyacrylamide gel, concentrated in the stacking gel, silver stained according to MS-compatible protocol, reduced, alkylated and digested with trypsin. Peptides were extracted from the gel with 0.1% trifluoroacetic acid and subjected to liquid chromatography. For peptide separation over a 140 minutes LC-gradient an Ultimate 3000 Rapid Separation liquid chromatography system (Dionex; ThermoFisher Scientific) equipped with an Acclaim PepMap 100 C18 column (75 μm inner diameter x 50 cm length x 2 mm particle size from ThermoFisher Scientific) was used. Mass spectrometry was carried out on an Obitrap Elite high-resolution instrument (ThermoFisher Scientific) operated in positive mode and equipped with a Nano electrospray ionization source. Capillary temperature was set to 275°C and source voltage to 1.5 kV. Survey scans were conducted in the orbitrap analyzer at a mass to charge (m/z) ranging from 350-1700 and a resolution of 60,000 (at 400 m/z). The target value for the automatic gain control was 1,000,000 and the maximum fill time 200 ms. The 20 most intense doubly and triply charged peptide ions (minimal signal intensity 500) were isolated, transferred to the linear ion trap (LTQ) part of the instrument and fragmented using collision induced dissociation (CID). Peptide fragments were analyzed using a maximal fill time of 200 ms and automatic gain control target value of 100,000. The available mass range was 200- 2000 m/z at a resolution of 5400 (at 400 m/z). Two fragment spectra were summed up and already fragmented ions excluded from fragmentation for 45 seconds.

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).