MassIVE MSV000089149

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Quantitative proteomics study of Sideroxydans lithotrophicus grown on solid and aqueous Fe (II)

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Description

The cells were grown on Fe(II)-citrate as the aqueous Fe(II) source and Fe(II)-smectite as the solid Fe(II) source. Cells were collected on a 0.2 um filter membrane (Millipore GPWP) by filtration. Then cells on the membrane were lysed by vortex in the lysis buffer containing 100 mM Tris/HCl, 10 mM Ethylenediaminetetraacetic acid (EDTA), pH 8.0, 0.05% Tween-20 and 1% w/v sodium dodecylsulfate (SDS). The proteins were digested with trypsin using in-house packed glass fiber filters following the suspension trapping (STrap) protocol, and desalted using C18-based StageTips. The LC-MS/MS analysis was performed using an Orbitrap Eclipse MS (Thermo Scientific) coupled with an Ultimate 3000 nanoLC system and a FAIMS Pro Interface (Thermo Scientific). Peptides were first loaded onto a trap column (PepMap C18; 2 cm x 100 um I.D.) and then separated by an analytical column (PepMap C18, 3.0 um; 10 cm x 75 um I.D.; Thermo Scientific) at 300 nl/min flow rate using a binary buffer system (buffer A, 0.1% formic acid in water; buffer B, 0.1% formic acid in acetonitrile) with a 165-min gradient (1% to 10% in 8 min; then to 25% buffer B over 117 min; 25% to 32% buffer B in 10 min, then to 95% buffer B over 3 min; back to 1% B in 5 min, and stay equilibration at 1% B for 20 min). Multiple CVs (-50, -65 and -80) were applied for FAIMS separation. For all experiments, the survey scans (MS1) were acquired over a mass range of 375-1500 m/z at a resolution of 60,000 in the Orbitrap. The maximum injection time was set to Auto, and AGC target was set to Standard. Monoisotopic peak selection was set to Peptides, and the charge state filter was set to 2-7. For MS/MS acquisition, precursors were isolated with a width of 1.6 m/z, fragmented with HCD using 30% collision energy with a maximum injection time of 100 ms, and collected in Orbitrap at 15,000 resolution. The dynamic exclusion was set to 60 s, and can be shared across different FAIMS experiments. Protein identification and quantitation were performed using the MaxQuant-Andromeda software suite (version 1.6.3.4) with most of the default parameters. An UniProt database (Sideroxydans lithotrophicus ES-1; Taxonomy ID, 580332; 2,978 sequences) was used for the protein identification. Other parameters include: trypsin as an enzyme with maximally two missed cleavage sites; protein N-terminal acetylation and methionine oxidation as variable modifications; cysteine carbamidomethylation as a fixed modification; peptide length must be at least 7 amino acids. False discovery rate was set at 1% for both proteins and peptides. Perseus (version 1.6.7.8) was used for downstream bioinformatics analysis such as clustering, correlation, t-test and ANOVA. [doi:10.25345/C55T3G370] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Label free quantitation ; Quantitative proteomics ; MaxQuant ; Sideroxydans lithotrophicus ; Iron

Contact

Principal Investigators:
(in alphabetical order) 		CLARA CHAN, School of Marine Science and Policy, University of Delaware, Newark, Delaware, USA, USA
Submitting User: yayu

Publications

Zhou N, Kupper RJ, Catalano JG, Thompson A, Chan CS.
Biological Oxidation of Fe(II)-Bearing Smectite by Microaerophilic Iron Oxidizer Sideroxydans lithotrophicus Using Dual Mto and Cyc2 Iron Oxidation Pathways.
Environ Sci Technol. 2022 Dec 6;56(23):17443-17453. Epub 2022 Nov 23.

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