DOI: 10.25345/c5rb6wg1k
title: MassIVE MSV000100331 - DIA analysis of Bone Marrow-Derived Macrophages
title: DIA analysis of Bone Marrow-Derived Macrophages Sample preparation Cell lysis and protein digestion. Bone marrow-derived macrophage (BMDM) cell pellets lysed in Urea buffer (8 M urea, 100 mM Tris, pH 8.0). Lysates were briefly homogenized by probe sonication, and protein concentrations were determined. Aliquots corresponding to 50 ug of total protein were transferred into new tubes and adjusted to a final concentration of 1 mg/mL with Urea buffer. Cysteine residues were reduced and alkylated by addition of a 10x stock solution containing TCEP (100 mM) and chloroacetamide (400 mM) in water, followed by incubation for 30 min at room temperature in the dark. The urea concentration was subsequently diluted to 1 M with 100 mM Tris, pH 8.0. Next, proteins were digested overnight at room temperature using Trypsin (sequencing-grade frozen trypsin, 0.4 ug/uL; Promega, V5113) and Lys-C (mass-spectrometry grade, 1 ug/uL in water; Wako Chemicals, 129-02541) at an enzyme-to-protein ratio of 1:50 (w/w) for each enzyme. Digestion was quenched by acidifying samples to a final concentration of 0.5% TFA. Peptide desalting. Digests were desalted using solid-phase extraction (SPE) cartridges (1 cc, 50 mg C18; Sep-Pak, Waters) operated on a vacuum manifold. Cartridges were conditioned with 100% acetonitrile (ACN), followed by two washes with 0.2% formic acid (FA). Acidified digests were then loaded onto the cartridges, washed twice with 0.2% FA, and eluted with 80% ACN containing 0.2% FA. Eluates were dried in a vacuum concentrator and resuspended in 0.2% FA for subsequent LC-MS analysis. LC-MS analysis For LC-MS analysis, a Vanquish Neo UHPLC system (Thermo Fisher Scientific) was coupled to an Orbitrap Astral mass spectrometer (Thermo Fisher Scientific) equipped with a Nanospray Flex ion source. Peptides were separated on a 40 cm in-house-packed fused-silica column (C18, 1.7 um, 130 A) maintained at 50 C using a custom-built column heater. The spray voltage was set to 2 kV, and the ion-transfer-tube temperature was held at 280 C. Peptides (250 ng total per injection) were separated at a flow rate of 300 nL/min using a 30-min active gradient from 5% to 46% solvent B (solvent A: 0.2% formic acid in water; solvent B: 80% acetonitrile with 0.2% formic acid). MS data were acquired in data-independent acquisition (DIA) mode on the Orbitrap Astral mass spectrometer (Thermo Fisher Scientific). MS1 acquisition. Full MS1 scans were recorded in the Orbitrap detector at a resolution of 240,000 over a scan range of 380-980 m/z. The maximum injection time was set to 50 ms, the normalized AGC target to 250%, and the RF lens to 40%. DIA acquisition. DIA scans were acquired in the Astral detector using an isolation window width of 4 m/z with no window overlap across a precursor mass range of 380-980 m/z. The DIA window type was set to Auto, and loop control was defined by time. Fragmentation was performed using higher-energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 25%. The MS2 scan range was 150-2000 m/z, the injection time was 2.5 ms, and the normalized AGC target was 500%. The RF lens was maintained at 40%. The total cycle time was 0.6 s, providing rapid duty cycles for comprehensive DIA coverage. Data analysis RAW files from DIA runs were analyzed in Spectronaut (version 19.9.250512.62635; Biognosys AG) using the directDIA+ (Deep) workflow. Unless otherwise specified, all parameters were kept at their default factory settings. The UniProt Mouse Reference Proteome (UP000000589; downloaded May 2025) was used as the search database. The following non-default settings were applied: Precursor PEP cutoff = 0.01, Protein Q-value cutoff (run level) = 0.01, Protein PEP cutoff = 0.01, and Exclude single-hit proteins = True. All other parameters, including FDR control, cross-run normalization, and interference correction, followed Spectronauts standard BGS Factory Settings. Protein group-level quantification data were exported from Spectronaut in pivot-transformed format. For each protein group, the number of valid PG.Quantity entries across all 32 samples was determined. Protein groups with fewer than 16 valid values were removed from further analysis. Remaining missing values were imputed in Perseus (version 2.1.3.0) using the Replace missing values from normal distribution function, with a Gaussian width of 0.3, a down shift of 1.8 standard deviations, and mode = separately for each column. Protein group-level quantification data were exported from Spectronaut in pivot-transformed format. For each protein group, the number of valid PG.Quantity entries across all 32 samples was determined. Protein groups with fewer than 16 valid values were removed from further analysis. Remaining missing values were imputed in Perseus (version 2.1.3.0) using the Replace missing values from normal distribution function, with a Gaussian width of 0.3, a down shift of 1.8 standard deviations, and mode = separately for each column.