assays/2_7-ProteomeAcquisition-MassSpectrometry/protocols/ProteomeAcquisition-MassSpectrometry.md

+# 2.7 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 an Ultimate 3000 Rapid Separation liquid chromatography system (Dionex; ThermoFisher Scientific) equipped with an Acclaim PepMap 100 C18 column (75 mm inner diameter x 50 cm length x 2 mm particle size from ThermoFisher Scientific) was used with a 140-minute LC- gradient. 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.
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