DOI: 10.6084/m9.figshare.24305218.v1
title: Regional differences in the small intestine proteome of control mice and of mice lacking lysosomal acid lipase

description: Despite recent studies investigating the involvement of single cells in regional differences of the small intestine (SI), the metabolic contribution of the duodenum, jejunum, and ileum to the overall intestinal metabolism is still unclear.Basic procedures and main findings: Using an untargeted proteomics approach, we identified similarities and differences between the three intestinal tracts of C57BL/6J mice and found that proteins highly abundant in the mouse ileum correlated with high ileal expression of the corresponding genes in humans. Consistent with human data, we detected increasing abundance of lysosomal acid lipase (LAL) in C57BL/6J mice from the duodenum to ileum. LAL is the only enzyme known to degrade triacylglycerols and cholesteryl esters within the lysosome. Lipid accumulation in various organs along with gastrointestinal disturbances and malabsorption are typical features of patients and mice with LAL deficiency. We previously demonstrated that macrophages massively infiltrate the SI of Lal-deficient (KO) mice, especially the duodenum. To identify potential mechanisms behind the intestinal lipid accumulation and infiltration of immune cells, we used untargeted proteomics. Our results revealed a general inflammatory response and a common lipid-associated macrophage phenotype in all three intestinal segments of Lal KO mice, accompanied by higher expression of GPNMB and increased concentrations of circulating sTREM2. However, only duodenal macrophages activated a metabolic switch from lipids to other pathways such as glycolysis, TCA cycle, and oxidative phosphorylation to meet their high energy demand. Unexpectedly, these pathways were downregulated in the jejunum and ileum of Lal KO mice.Conclusion: Our results provide new insights into the process of absorption in control mice and the basis for novel markers of LAL-D and/or systemic inflammation in LAL-D.<br>Sample Processing protocol:Duodena, jejuna, and ilea from 6-h fasted male WT and Lal KO mice (n=6/group, 30-33 weeks of age) were pooled into groups of two and lysed with 8 M urea, 0.1 M Tris-HCl (pH 8.5) in the presence of protease inhibitors (# 5872S, 1:100; Cell Signaling, Technology, Danvers, MA) for 30 min at 4 °C with constant shaking. Samples were then centrifuged at 14,000 x g and 4 °C for 30 min. The supernatant was collected and proteins were quantified using the Lowry protein assay. A total of 10 µg of proteins were completely dried in a vacuum concentrator at 45 °C for 45 min. Afterwards, the dried protein pellet was resuspended in 10 µl of water plus 10 µl of 50 mM ammonium bicarbonate solution (final pH 8.5), followed by protein reduction with 5 mM DTT for 20 min at 55 °C as previously described [18,19]. Proteins were then alkylated at room temperature by incubation with 15 mM iodoacetamide for 30 min in the dark. Trypsin (#T7575-1KT, Merck Millipore, Billerica, MA) digestion with a 1:20 ratio of enzyme to protein was performed overnight at 37 °C and terminated by acidification with trifluoroacetic acid (final concentration 1%). Proteolytic peptide mixtures were purified by C18 resin pipette tips and analyzed in duplicate using Dionex Ultimate 3000 nano-LC system (Sunnyvale, CA) connected to an Orbitrap Tribrid mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with nano-electrospray ion source using mobile phase and elution gradient as previously described. Full MS scans were collected in positive ion mode at a resolution of 120,000 (375 to 1500 m/z range), operating in the data-dependent mode, cycle time 3s between master scans. MS/MS spectra were collected in centroid mode.<br>Data processing protocol :Data were processed and analyzed as previously described. Briefly, the raw MS data files were transformed to mzML format (MSconvert tool of the ProteoWizard program, version 3.0.1957; Palo Alto, CA, US). MzML files were then analyzed using OpenMS (version 2.64, deNBI, Germany) nodes running on the open-source software platform KNIME® (version 4.6, Knime AG, Zurich, Switzerland). MS/MS spectra were searched against a mouse Uniprot FASTA database (uniprot-mus+musculus.fasta, downloaded from www.uniprot.org, Jan 2022, 17.527 entries) and a common contaminant protein database using the DDASSQ pipeline as previously described. The OpenMS PeptideIndexer node was used to index peptide sequences with a defined leucine/isoleucine equivalence. The Protein Inference Analysis was then applied to infer proteins using the default parameters provided by the developers. Estimates of protein abundance were determined using the FeatureFinderMultiplex node to generate spectral features. Thereafter, Protein Inference Analysis -assisted false discovery rate (FDR)-multiple score estimation and filtering (combined FDR score 0.01), ID mapping and combination with peptide IDs, and subsequent alignment, grouping, and normalization (i.e. MapAlignerIdentification, FeatureUnlabeledQT, and ConsensusmapNormalizer nodes) were performed. Next, the OpenMS ProteinQuantifier node was employed to calculate label-free quantification (LFQ) of proteins and peptides based on the intensities of the three most abundantly detected peptides.