MassIVE MSV000094145

Partial Public

Disrupted Nitric Oxide Homeostasis Impacts Fertility Through Multiple Processes Including Protein Quality Control

Description

Pistils of hot5-2 and WT at stages before pollination (FD 12-13) were harvested and immediately shock frozen in lN2. Around 50 pistils per genotype were pooled and represent one biological replicate with a total of three biological replicates per genotype. Proteins were extracted using diluted HENS buffer (25mM HEPES pH 7.7, 1mM EDTA, 2.5 % SDS, protease inhibitor cocktail (Pierce A32955), TCA precipitated and resuspended in the same buffer. After BCA protein determination, 50 ug of each whole cell lysate was run on a 4-20% SDS-PAGE for approx. 15 min to separate proteins from lower molecular weight contaminants. The entire protein region was excised of the gel and subjected to in-gel trypsin digestion after reduction with dithiothreitol and alkylation with iodoacetamide as described in (Treffon et al., 2021). Peptides eluted from the gel were lyophilized and re-suspended in 50uL of 5% acetonitrile (0.1% (v/v) FA). For each biological replicate, three technical replicates were measured by LC-MS/MS. A 2 uL injection was loaded by a Waters NanoAcquity UPLC in 5% acetonitrile (0.1% formic acid) at 4.0 uL/min for 4.0 min onto a 100 um I.D. fused-silica pre-column packed with 2 cm of 5 um (200A) Magic C18AQ (Bruker-Michrom). Peptides were eluted at 300 nL/min from a 75 um I.D. gravity-pulled analytical column packed with 25 cm of 3 um (100A) Magic C18AQ particles using a linear gradient from 5-35% of mobile phase B (acetonitrile + 0.1% formic acid) in mobile phase A (water + 0.1% formic acid) over 90 minutes. Ions were introduced by positive electrospray ionization via liquid junction at 1.4kV into a Thermo Scientific Q Exactive hybrid mass spectrometer. Mass spectra were acquired over m/z 300-1750 at 70,000 resolution (m/z 200) with an AGC target of 1e6, and data-dependent acquisition selected the top 10 most abundant precursor ions for tandem mass spectrometry by HCD fragmentation using an isolation width of 1.6 Da, max fill time of 110ms, and AGC target of 1e5. Peptides were fragmented by a normalized collisional energy of 27, and fragment spectra acquired at a resolution of 17,500 (m/z 200). [doi:10.25345/C5NZ8116V] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: gsnor/hot5_2 quantitative pistil proteome, AT5G43940

Contact

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