DOI: 10.13130/rd_unimi/sd7n5g
title: Replication Data for: Astrocytic ATM deficiency triggers complement C3a-mediated synaptic dysfunction and depressive-like behaviors

description: This dataset includes LC-MS/MS data of differentially expressed proteins in the astrocyte proteome and secretome generated in the study entitled “Astrocytic ATM deficiency triggers complement C3a–mediated synaptic dysfunction and depressive-like behaviors” by Sabrina Briguglio, Anna Selimi, Clara Cambria, Ramona Stringhi, Manuela Moriggi, Mario Federici, Maria Vittoria Zavaglia, Elena Borroni, Francesco Rusconi, Elena Battaglioli, Elena Marcello, Laura Musazzi, Daniele Capitanio, and Flavia Antonucci. 
Astrocytes were gently scraped, collected in PBS and pelletted. Astrocytes were then 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) was then performed. Culture media (15 ml) were centrifuged in 30 KDa MWCO filters (ThermoFisher scientific) to a final volume of 50 µl. Lysis buffer (50 µl) was then added, and each medium was processed as described for cells. Protein extracts (50 µg for each sample) were processed following the filter-aided sample preparation (FASP) protocol. Peptide samples were concentrated and then separated on a Dionex UltiMate 3000 HPLC System with an Easy Spray PepMap RSLC C18 column (250 mm, internal diameter of 75 µm) (ThermoFisher Scientific), and finally electrosprayed into an Orbitrap Fusion Tribrid (ThermoFisher Scientific) mass spectrometer. Three technical replicates for each sample were acquired.
Mass spectra were analysed 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. False positives were reduced utilizing the Benjamini–Hochberg false discovery rate test.
description: MaxQuant (Max Planck Institute of Biochemistry), 1.6.17.0
description: Perseus (Max Planck Institute of Biochemistry), 1.6.15.0
description: The related publication has not been published yet because it is still under review.