DOI: 10.25345/c5p55ds51
title: MassIVE MSV000091600 - GJN lung proteomics reveals insights into cellular signaling
title: For LC-MS/MS measurements, protein samples (20 ug/lane) were first submitted to SDS-PAGE. The gel was then stained with Bio-SafeTM Coomassie stain (Bio-Rad), and de-stained with water. Then, protein bands corresponding to the molecular weight of the protein of interest were cut from the polyacrylamide gel and the gel fragments were digested using an in-gel tryptic digestion kit (Thermo Scientific: p/n 89871). Following digestion, extracted peptides were then desalted using PierceTM pipette tips (ThermoFisher) and dried, resuspended in 20 uL of 0.5% acetic acid (pH4.5) and then subjected to LC-MS/MS using an Thermo Scientific Orbitrap Eclipse Tribrid Mass Spectrometer (MS; Thermo Scientific) with an Ultimate 3000 nano-liquid chromatography (nano-LC) and a FAIMS Pro Interface (Thermo Scientific). The LC-MS/MS analysis was performed using an Orbitrap Eclipse MS (Thermo Scientific). Peptides were first loaded onto a trap column (PepMap C18) and then separated by an analytical column (PepMap C18, 2.0 um; 15 cm x 75mm 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 (-40, -55 and -75) 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 Dynamic, 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. LC-MS/MS data was collected in n=3 independent biological replicates. Proteomic analysis was performed in the MaxQuant-Andromeda software suite (version 1.6.3.4) with most of the default parameters51. An E. coli reference proteome (strain BL21-DE3; taxonomy_id:469008) was used for database search. Other parameters include: trypsin as an enzyme with maximally two missed cleavage sites; protein N-terminal acetylation, methionine oxidation, and pA-Phe and pN-Phe substitution for tyrosine were entered as variable modifications; cysteine carbamidomethylation as a fixed modification; and peptide length was set to a minimum of 7 amino acids. The false-discovery rate of high-confidence protein and peptide identification was 1%. Peptide intensity values derived from MaxQuant were used for quantification. To obtain the MS/MS spectra of peptides, additional analysis was performed using Proteome Discoverer software (version 3.0; Thermo Fisher). Raw data was searched against the E. coli reference proteome (strain BL21(DE3)) with NCBI reference Proteome (469008) using Sequest HT Processor and CWF Basic analysis workflows. Iodoacetamide-mediated cysteine carbamidomethylation was set as a static modification, while methionine oxidation and pA-Phe and pN-Phe substitution for tyrosine were entered as dynamic modifications. Precursor mass tolerance was set at 10ppm while allowing fragment ions to have a mass deviation of 0.02?Da for the HCD data. Validation of peptide-spectrum matches (PSM) based on q value was done using Percolator, with target false-discovery rates (FDR) of 1% and 5% for stringent and relaxed validations, respectively.