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Differential proteomic analysis reveals the mechanism of Providencia.sp to selenium and manganese stress

ABSTRACT: In this study, iTRAQ technique is employed to reveal the proteomic response of Providencia. sp under selenium and manganese stress. After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried. Peptide was reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for TMT kit/iTRAQ kit. Then, The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Agilent 300Extend C18 column. Finally, The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC.

ORGANISM(S): Providencia Rettgeri 947

SUBMITTER: LiFang wu

PROVIDER: PXD022749 | iProX | Thu Nov 26 00:00:00 GMT 2020

REPOSITORIES: iProX

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Project description:For proteomics analysis, testes of Ube2j2-/- and WT mice were collected on ice, washed once with pre-cooled PBS, dried with absorbent paper, and immediately placed in liquid nitrogen, dry ice, or at -80 ℃. Samples were packed in dry ice and transported to Hangzhou Jingjie Biotechnology Company (PTM Biolabs, Inc.) for whole protein 4D label-free quantitative proteomic determination. Testes of Ube2j2-/- and WT mice were subjected to LC-MS/MS analysis following a standard protocol. The sample was grinded and mixed with lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail III (Merck Millipore, 539134) dissolved in aqueous solution containing buffer salts), followed by sonication on ice using a high intensity ultrasonic processor (Scientz). The remaining debris was removed by centrifugation at 12,000 g at 4 °C for 10 min. Then, the supernatant was collected and the protein concentration was determined with BCA kit (Beyotime Biotechnology, P0011) according to the manufacturer’s instructions. The sample was slowly added to a final concentration of 20% (m/v) trichloroacetic acid (Sigma-Aldrich, T4885) to precipitate proteins, then mixed by vortexing and incubated for 2 h at 4 °C. The precipitate was collected by centrifugation at 4500 x g for 5 min at 4°C. The precipitated protein was washed 3 times with pre-cooled acetone and dried for 1 min. Protein samples were then redissolved in 200 mM TEAB (Triethylammonium bicarbonate buffer, Sigma-Aldrich, T7408) and ultrasonically dispersed. Trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight. The sample was reduced with 5 mM dithiothreitol for 60 min at 37 °C and alkylated with 11 mM iodoacetamide for 45 min at room temperature in darkness. Finally, the peptides were desalted using a Strata X SPE column (Phenomenex). The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Agilent 300Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). The released peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. Maxquant 1.6.15.0 software was used for spectra analysis, database searches, and peptide identification.

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Differential proteomic analysis reveals the mechanism of Providencia.sp to selenium and manganese stress

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