69 3 Responses of alveolar-like macrophages to lysine deacetylase inhibition proteomics of metabolic enzymes by QconCATs” were analyzed. Untargeted proteome analyses were performed by injecting 1 µg total protein starting material on a quadrupole orbitrap mass spectrometer equipped with a nano-electrospray ion source (Orbitrap Exploris 480, Thermo Scientific). Chromatographic separation of the peptides was performed by liquid chromatography (LC) on an Evosep system (Evosep One, Evosep) using a nano-LC column (EV1137 Performance column 15 cm x 150 µm, 1.5 µm, Evosep; buffer A: 0.1% v/v formic acid, dissolved in milliQ-H2O, buffer B: 0.1% v/v formic acid, dissolved in acetonitrile). Peptides were separated using the 30SPD workflow (Evosep). The mass spectrometer was operated in positive ion mode and data-independent acquisition mode (DIA) using isolation windows of 16 m/z with a precursor mass range of 400-1000, switching the high-field asymmetric waveform ion mobility spectrometry (FAIMS) between CV-45V and -60V with three scheduled MS1 scans during each screening of the precursor mass range. LC-MS raw data were processed with Spectronaut (version 16.0.220606) (Biognosys) using the standard settings of the directDIA workflow except that quantification was performed on MS1, with a mouse Swissprot database (17021 protein entries, downloaded in December 2019). For the quantification, local normalization was applied and the Q-value filtering was set to the classic setting without imputing. Statistical analysis Analyses were performed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, USA). Due to the limited sample size in our experiments, nonparametric testing was used to compare groups. For nonparametric testing between two groups a Mann-Whitney U test was used for unpaired or a Wilcoxon test for paired data. For comparison of multiple groups, a Kruskall Wallis or Friedman test was used for nonpaired or paired nonparametric data, respectively, with Dunn’s correction for multiple testing. Proteomics data were normalized using Local Normalization based on the Local Regression Normalization described by Callister et al. (32) and Log2 transformed. Groups were subsequently compared using a paired one-way ANOVA with Holm-Sidak’s correction for multiple testing. When comparing lists of proteins fold changes and p-values adjusted for multiple testing (q values) were generated. Differentially expressed protein were subsequently selected having q-values <0.05. Data Availability The untargeted mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (33) with the dataset identifier PXD040933. The targeted mass spectrometry proteomics data
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