Computational protocol: Scale Invariant Disordered Nanotopography Promotes Hippocampal Neuron Development and Maturation with Involvement of Mechanotransductive Pathways

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[…] The cells interacted for 3 days with the indicated substrates (in total 4 coverslips with Ø13 mm each, representing 5.3 cm2 cumulative substrate area). Then the cells were scratched from the substrates with a cell scraper (TPP, Trasadingen, Switzerland) (on ice) in the presence of icecold PBS supplemented with protease inhibitors (Roche, Basel, Switzerland).After reduction and derivatization, the proteins were digested with trypsin sequence grade trypsin (Roche) for 16 h at 37°C using a protein:trypsin ratio of 1:50. LC-ESI-MS/MS analysis was performed on a Dionex UltiMate 3000 HPLC System with a PicoFrit ProteoPrep C18 column (200 mm, internal diameter of 75 μm) (New Objective, USA). Gradient: 1% ACN in 0.1% formic acid for 10 min, 1–4% ACN in 0.1% formic acid for 6 min, 4–30% ACN in 0.1% formic acid for 147 min and 30–50% ACN in 0.1% formic for 3 min at a flow rate of 0.3 μl/min. The eluate was electrosprayed into an LTQ Orbitrap Velos (Thermo Fisher Scientific) through a Proxeon nanoelectrospray ion source (Thermo Fisher Scientific). The LTQ-Orbitrap was operated in positive mode in data-dependent acquisition mode to automatically alternate between a full scan (m/z 350–2000) in the Orbitrap (at resolution 60000, AGC target 1000000) and subsequent CID MS/MS in the linear ion trap of the 20 most intense peaks from full scan (normalized collision energy of 35%, 10 ms activation). Isolation window: 3 Da, unassigned charge states: Rejected, charge state 1: Rejected, charge states 2+, 3+, 4+: Not rejected; dynamic exclusion enabled (60 s, exclusion list size: 200). Five technical replicate analyses of each sample were performed. Data acquisition was controlled by Xcalibur 2.0 and Tune 2.4 software (Thermo Fisher Scientific) (Aletti et al., ).The mass spectra were analyzed using MaxQuant software (version (Cox and Mann, ). The initial maximum allowed mass deviation was set to 6 ppm for monoisotopic precursor ions and 0.5 Da for MS/MS peaks. The enzyme specificity was set to trypsin, defined as C-terminal to arginine and lysine excluding proline, and a maximum of two missed cleavages were allowed. Carbamidomethylcysteine was set as a fixed modification, N-terminal acetylation, methionine oxidation and serine/threonine/tyrosine phosphorylation as variable modifications. The spectra were searched by the Andromeda search engine against the rat Uniprot sequence database (release 04.07.2014) and the mouse Uniprot sequence database (release 04.07.2014). The reversed sequences of the target database were used as decoy database. Protein identification required at least one unique or razor peptide per protein group. The quantification in MaxQuant was performed using the built-in XIC-based label free quantification (LFQ) algorithm using fast LFQ (Cox and Mann, ). The required false positive rate was set to 1% at the peptide and 1% at the protein level against a concatenated target decoy database, and the minimum required peptide length was set to 6 amino acids. Statistical analyses were performed using the Perseus software (version, Only proteins present and quantified in at least 3 out of 5 technical repeats were considered as positively identified in a sample and used for statistical analyses. An ANOVA test (false discovery rate 0.05) was carried out to identify proteins differentially expressed among the three conditions.We performed the comparison between cells grown on nanostructured zirconia with the roughness Rq of 25 nm rms and the flat surfaces; i.e., Control (glass coverslips) and flat-Zr, in order to better understand the effect of the surface nanotopography. Common proteins were considered to be differentially expressed if they were present only in Control, flat-Zr, or the ns-Zr25 or showed a significant t-test difference (cut-off at 5% permutation-based False Discovery Rate). These proteins were filtered for further analyses. Proteins known to be due to a contamination of the matrigel were excluded from the analysis.The differently expressed proteins were clustered according to their functions using the Panther platform (Version 10.0 release date April 25, 2015) (Mi et al., ) and filtered for significant Gene Ontology terms: Biological Process (GO-SlimBP) and Pathways using a p value < 0.05.Genuine mitochondrial protein localization was determined by Mitominer, a database of the mitochondrial proteome which integrates protein data from HomoloGene, Gene Ontology, KEGG, OMIM MS/MS, GFP (green fluorescent protein) localization data and targeting sequence predictions. Only proteins with an Integrated Mitochondrial Protein Index (IMPI) ≥ 0.5 were considered true mitochondrial molecules (Smith et al., ). […]

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