DOI: 10.25345/c50863g9d
title: MassIVE MSV000093770 - Mechanisms of extracellular electron transport in Sideroxydans lithotrophicus ES-1
title: The ES-1 cells were processed following the E4technology protocol described recently. In brief, the fresh cells collected onto the E4membranes were first rinsed two times with 2.0 ml cold phosphate-buffered saline (PBS) to deplete residual culturing media. The cells were then washed with 1.0 ml pure methanol once, and then incubated on ice with 1.0 ml methanol for 60 min. After centrifugation to discard the supernatant, the cells were added 200 ul of 50 mM triethylammonium bicarbonate (TEAB) and 1.0 ug of trypsin (Promega, Madison, WI). The samples were incubated at 37 C for 16-18 hours. Afterwards, the samples on the filters were first eluted with 200 ul of 0.2% formic acid (FA) in waters and then 200 ul of 0.2% formic acid in 50% ACN/water by spinning at 2,000 rpm for 1-2 min. The elution was pooled and dried in SpeedVac. The dried peptides were reduced and alkylated with 10 mM TCEP and 40 mM CAA in 50 mM TEAB by incubating at 37 C for 20 min. The reaction was stopped by adding 200 ul 1% TFA in water. The samples were then desalted following the C18 StageTip protocol. For LC-MS/MS analysis, peptides were resuspended in 20 ul of LC buffer A (0.1% FA in water), and then were separated at 250 nl/min on an Ultimate 3000 RSLCnano system coupled with a trap column (PepMap100 C18, 300 um x 2 mm, 5 um; Thermo Scientific) and an analytical column (PepMap100 C18, 25 cm x 75 um i.d., 3 um; Thermo Scientific). A linear LC gradient was applied from 1% to 25% mobile phase B over 125 minutes, followed by an increase to 32% mobile phase B over 10 minutes. The column was washed with 95% mobile phase B for 5 minutes, followed by equilibration with mobile phase A for 15 minutes. The spectra were collected on an Orbitrap Eclipse system installed with FAIMS Pro Interface (Thermo Scientific). For FAIMS acquisition, a 3-CV experiment (-40|-55|-75) was applied. For the ion source, the spray voltage was set to 1.7 kV, funnel RF level at 50%, and heated capillary temperature at 275 C. The MS data were acquired by Orbitrap at 60,000 resolution, followed by MS/MS acquisition of the most intense precursors for 1 s. The MS1 scan range was set to 375-1500 m/z, AGC target was set to Standard, and the maximum injection time mode was set to 50 milli seconds. For MS2 analysis, precursor with charge states 2-5 were selected. The isolation mode was Quadrupole, collision was by HCD at 30% normalized collision energy (NCE). The Orbitrap was set to detect MS2 fragments at 15,000 resolution with standard AGC target, Dynamic maximum injection time, and a 1.6 m/z isolation window. Dynamic exclusion was set to 30 s. Monoisotopic precursor selection (MIPS) was set to Peptide. For proteome quantitation, raw MS data were processed using MaxQuant and Andromeda software suite (version 2.4.2.0). Protein databases of Sideroxydans lithotrophicus ES-1 cells (taxonomy_id:580332; 2,978 sequences) was downloaded from UniProtKB website. The enzyme specificity was set to Trypsin; variable modifications include oxidation of methionine, and acetyl (protein N-terminus); fixed modification includes carbamidomethylation of cysteine. The maximum missed cleavage sites were set to 2 and the minimum number of amino acids required for peptide identification was 7. The false discovery rate (FDR) was set to 1% for protein and peptide identifications. MaxLFQ function embedded in MaxQuant was enabled for label-free quantitation, and the LFQ minimum ratio count set to 1. Proteins identified as reverse hits, potential contaminants, or only by site-modification were filtered out from the proteinGroups.txt output file. All the statistical analyses, including histograms, Pearson correlation, heatmap, volcano plot, and t-tests, were performed using either Perseus (version 1.6.2.3) or GraphPad Prism (version 9.5.1) if not indicated. The LFQ values were log2 transformed, filtered by at least two valid values out of three replicates, imputed using the default normal distribution method in Perseus.