Dataset Information

Data-Independent Acquisition proteomics of protein extracted from kernels collected at fifteen and thirty days post self-pollination of the ubp5 mutant heterozygous (smd/+) and wild-type specimens

ABSTRACT: protein extraction was performed from kernels collected at fifteen and thirty days post self-pollination of the ubp5 mutant heterozygous (smd/+) and wild-type specimens using phenol. For quantifying protein expression levels, 500μg of dissolved protein underwent reduction by 50 mM DTT (3483-12-3, Sigma-Aldrich) at 37°C for 1 hour. Subsequently, 150 mM iodoacetamide (144-48-9, Sigma-Aldrich) was added to modify the reduced sulfhydryl groups. Following a 30-minute incubation period in darkness at room temperature, the protein sample underwent buffer exchange to 50 mM ammonium bicarbonate (A6141, Sigma-Aldrich) through an ultrafiltration column (UFC501096, Millipore). Enzymatic digestion was carried out using trypsin (V5111, Promega), and the resulting digest underwent purification using C18 Spin Tips (84850, Thermo) followed by elution with 60% acetonitrile and rapid drying under vacuum conditions.

ORGANISM(S): Zea Mays

SUBMITTER: Zecheng Zuo

PROVIDER: PXD047889 | iProX | Sun Dec 17 00:00:00 GMT 2023

REPOSITORIES: iProX

ACCESS DATA

Similar Datasets

Project description:Human keratinocytes, HaCaT cells were transfected with the dsRNA-analogue polyinosinic-polycytidylic acid (poly I:C) (Sigma-Aldrich) using Lipofectamine 2000 reagent (Invitrogen) for intracellular delivery. The cells were transfected with 7 ug/ml poly I:C for 1 h or left untreated. The cells were collected in PBS with a cell scraper and washed once with PBS before they were lysed with HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, pH 7.4) including protease and phosphatase inhibitor cocktails (Sigma-Aldrich). The cell lysates were centrifuged 2,264 x g for 15 min at 4°C, divided into smaller fractions and centrifuged again 11,686 x g for 15 min at 4°C . The supernatant was collected and the protein content was measured with Bio-Rad DC protein assay (Bio-Rad). For the samples, 8 mg of protein was used. The proteins were precipitated with 10 % TCA/acetone at -20°C overnight. Samples were centrifuged 10,733 x g for 20 min at 4°C and the supernatant was removed. The precipitation was washed once with acetone and dissolved in urea buffer (8 M urea, 400 mM NH4CO3, 20 mM DL-dithiotheitol, 1 mM EDTA, pH 8.5). The proteins were reduced, alkylated, and enzymatically digested in-solution with lysyl endopeptidase (Wako Chemicals) and trypsin (Promega). The peptides were fractionated by strong cation exchange chromatography. Phosphopeptide enrichment was performed with immobilized-metal affinity chromatography (IMAC) using PHOS-Select Iron Affinity Gel (Sigma Aldrich). The enriched phosphopeptides were analyzed by nanoLC-MS/MS using an Ultimate 3000 nano-LC (Dionex) coupled to a QSTAR Elite hybrid quadrupole TOF-MS (Applied Biosystems/MDS Sciex) with nano-ESI ionization.23,24 The LC-MS/MS data were submitted through the ProteinPilot 4.0 interface (Applied Biosystems/MDS Sciex) to an in-house Mascot database search engine version 4.0 (Matrix Science), and to the ProteinPilot algorithm Paragon. The data were searched against the human canonical sequences in the Swiss-Prot database (decoy version 08132012 for the Mascot searches and version 04012013 with 539,829 sequences for the Paragon searches). The Mascot search criteria were: fixed modifications: carbamidomethyl of cysteine, variable modifications: oxidation (M), phospho (ST), phospho (Y), acetylation (K), 1 missed cleavage allowed, enzyme: trypsin. The Paragon search criteria were: modifications: cystein alkylation with iodoacetamide, ID focus: biological modifications, phosphorylation emphasis, identification threshold: 95 % confidence (Unused ProtScore (Conf) > 1.3), Thorough search (ID), enzyme: trypsin.

Dataset Information

Data-Independent Acquisition proteomics of protein extracted from kernels collected at fifteen and thirty days post self-pollination of the ubp5 mutant heterozygous (smd/+) and wild-type specimens

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure