Replication Data for: Post-Weaning Microbiota Colonization in Germ-Free Mice Compensates for Impaired Skeletal Muscle Maturation but Not for Peripheral Nerve Fiber Abnormalities

This dataset includes LC-MS/MS data of differentially expressed proteins in the proteome generated in the study entitled “Post-Weaning Microbiota Colonization in Germ-Free Mice Compensates for Impaired Skeletal Muscle Maturation but Not for Peripheral Nerve Fiber Abnormalities” by Sonia Calabrò, Davide Pellegrino, Chiara Cicconetti, Svenja Kankowski, Sajjad Farzin, Francesca Bertone, Mira Frühwein, Francesca Anselmi, Dario Bizzotto, Marijana Basic, Silvia Bolsega, Matthias Steglich, Lutz Wiehlmann, Cecilia Gelfi, Daniele Capitanio, Karin Kleigrewe, Salvatore Oliviero, Giovanna Gambarotta, Kirsten Haastert-Talini, Matilde Cescon, and Giulia Ronchi.
Gastrocnemius muscle biopsies were suspended in lysis buffer (2% SDS, 100 mM Tris-HCl pH 7.6, 0.1 M DTT, and 1% phenylmethanesulfonylfluoride) and sonicated on ice until completely dissolved. After incubation at 95 °C for 3 min, lysates were clarified by centrifugation at 12,000× g for 20 min at 4 °C. Protein quantitation with 2-D Quant-kit protein assay (Cytiva, Little Chalfont, UK) was then performed. Protein extracts (100 µg for each sample) were processed following the filter-aided sample preparation (FASP) protocol. Each sample was deposited in a Microcon-30 kDa centrifugal filter unit (Merck Millipore, Burlington, MA, USA) and washed by centrifugation at 14,000 g for 15 min with 200 µL of UA buffer (8 M urea, 0.1 M Tris/HCl, pH 8.5). Samples were carbamydomethylated in 100 µL of 50 mM iodoacetamide in UA buffer for 20 min, then washed three times in 100 µL UA buffer followed by three washes in 100 µL of 50 mM ammonium bicarbonate in water. Filters were incubated with sequence grade trypsin (Promega, Madison, WI, USA) for 16 h at 37 °C using a protein:trypsin ratio of 50:1. After acidification with trifluoracetic acid and desalting on C18 tips (Zip-Tip C18 micro, Merck Millipore, Burlington, MA, USA), peptide samples were vacuum concentrated, reconstituted in HPLC buffer A (0.1% formic acid) and separated on a Dionex UltiMate 3000 HPLC System with an Easy Spray PepMap RSLC C18 column (250 mm, internal diameter of 75 µm) (Thermo Fisher Scientific), and electrosprayed into an Orbitrap Fusion Tribrid (Thermo Fisher Scientific) mass spectrometer. Three technical replicates for each sample were acquired. Mass spectra were analyzed using MaxQuant software (Max Planck Institute of Biochemistry, Munich, Germany, version 1.6.17.0). The maximum allowed mass deviation was set to 6 ppm for monoisotopic precursor ions and 0.5 Da for MS/MS peaks. Enzyme specificity was set to trypsin/P, and a maximum of two missed cleavages was allowed. Carbamidomethylation was set as a fixed modification, while N-terminal acetylation and methionine oxidation were set as variable modifications. Spectra were searched by the Andromeda search engine against the Mus musculus Uniprot UP000000589 sequence database (54707 proteins, March 2024). Protein identification required at least one unique or razor peptide per protein group. Quantification in MaxQuant was performed using the built-in extracted ion chromatogram (XIC)-based label-free quantification (LFQ) algorithm using fast LFQ. The required FDR was set to 1% at the peptide, 1% at the protein, and 1% at the site-modification levels, and the minimum required peptide length was set to 7 amino acids. Statistical analyses were performed using Perseus software (v.1.6.15.0, Max Planck Institute of Biochemistry, Martinsried, Germany). For each experimental group, proteins identified in at least 80% of samples were considered. For statistical analysis, an ANOVA test with a p-value threshold of 0.05 was applied. False positives were reduced utilizing the Benjamini–Hochberg false discovery rate test.