MassIVE MSV000088017

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Quantitative proteome profiling of a S-Nitrosoglutathione reductase (GSNOR) null mutant reveals a new class of enzymes involved in nitric oxide homeostasis in plants

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Description

Leaves from 4 to 6 week old soil grown WT Col 0 and gsnor1/hot5_2 plants were used for protein extraction. For that, 200mg of plant material was resuspended in extraction buffer (25mM HEPES pH 7.7, 1mM EDTA, 2.5% [w/v] SDS, protease inhibitor cocktail (Pierce A32955)). Following TCA precipitation, proteins were resuspended in extraction buffer and protein concentration was determined using the BCA assay. 50ug of total protein per sample was run for 15min on an SDS-PAGE system to separate proteins from lower molecular weight contaminants, and the entire protein region of the gel excised and subjected to in-gel trypsin digestion after reduction with dithiothreitol and alkylation with iodoacetamide. Peptides eluted from the gel were lyophilized and re-suspended in 20 uL of 0.1% (v/v) FA. A 3uL injection was loaded by a Thermo Easy nLC 1000 UPLC onto a 2 cm trapping column and desalted with 8uL mobile phase A (0.1% formic acid in water). Peptides were then eluted at 300nL/min onto a 75 um x 50 cm RSLC column (Thermo) using a linear gradient of 5-35% mobile phase B (0.1% formic acid in acetonitrile) over 150 min. Ions were introduced by positive ESI using a stainless-steel capillary at 2.1 kV into a Thermo Orbitrap Fusion tribrid mass spectrometer. Mass spectra were acquired over m/z 300-1750 at 120,000 resolution (m/z 200) with an AGC target of 1e6, a cycle time of 1 s, and data-dependent acquisition at top speed selecting the most abundant precursor ions for tandem mass spectrometry by HCD fragmentation using an isolation width of 1.6 Da, maximum fill time 110ms and AGC target 1e5. Peptides were fragmented with a normalized collision energy 27, and fragment ion spectra acquired at turbo speed in the linear ion trap. Dynamic exclusion was applied with an exclusion of 15s after observing the same ion twice within 15s. Label-free quantification was performed with the MaxLFQ algorithm (Cox et al., 2014) using a minimum ratio count of 2. [doi:10.25345/C5MG0X] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: gsnor/hot5_2 quantitative leaf proteome, AT5G43940

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Principal Investigators:
(in alphabetical order) 		Elizabeth Vierling, University of Massachusetts Amherst, USA
Submitting User: PatrickTreffon
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