LC-MS/MS raw dataSpectrum matching and protein identification and validation were performed with MSFragger, and quantification of protein intensities with matching between runs was performed with IonQuant as components of the FragPipe analysis pipeline using the default settings of each module. The protein database used for the search was the Mus musculus reviewed sequence database downloaded from UniProt on June 1, 2023. The results were subsequently processed to filter out common contaminants, decoy hits from the reverse database, and protein groups identified by a single peptide. The data were filtered as follows: (a) binary expression of a protein (i.e., protein exclusively identified in either scLRP1+/+ or scLRP1-/-) was only considered relevant if all scLRP1+/+ samples or all scLRP1-/- samples expressed the protein. The missing values were imputed with the minimum intensity value for each specific data set; (b) for samples expressed in both scLRP1+/+ and scLRP1-/- tissue, the filtering process required 2 or more proteins to be detected in both scLRP1+/+ and scLRP1-/- samples. False discovery analysis was performed using the Benjamini, Krieger, and Yekutieli method using GraphPad Prism 10.0 software. Causal analysis of proteomic data was performed in IPA upstream analysis software (QIAGEN). For IPA, the binary values were imputed using local minimum intensities. Enrichment analyses for gene ontology (biological process) were performed using clusterProfiler 4.2.2 R package on R 4.1.0. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD038236. Global quantification of protein expression. Sciatic nerves from scLRP1+/+ and scLRP1-/- mice were rinsed in PBS to remove the blood and frozen with liquid nitrogen in cryogenic storage tubes (#5016-0001, Thermo Fisher Scientific). Fractured tissue was transferred to a 1 mL milliTUBE containing an AFA fiber (#520135, Covaris catalog) in 200 μL of 50 mM HEPES pH 8.5, 150 mM NaCl, and 2% Triton X-114 and sonicated with a M220 Focused-Ultrasonicator (#500295, Covaris catalog). Sonication parameters were temperature 15 °C, peak power 75 W, duty factor 26, cycles/burst = 1,000, and duration 600 seconds. Extracted proteins were clarified of insoluble material by centrifugation at 15,000 x g for 20 minutes at 4 °C. Protein concentrations were determined with the Micro BCA colorimetric assay (Pierce,...
Raw LC-MS/MS and RNA-Seq Mitochondria data
Stefano Martellucci;
2025-01-01
Abstract
LC-MS/MS raw dataSpectrum matching and protein identification and validation were performed with MSFragger, and quantification of protein intensities with matching between runs was performed with IonQuant as components of the FragPipe analysis pipeline using the default settings of each module. The protein database used for the search was the Mus musculus reviewed sequence database downloaded from UniProt on June 1, 2023. The results were subsequently processed to filter out common contaminants, decoy hits from the reverse database, and protein groups identified by a single peptide. The data were filtered as follows: (a) binary expression of a protein (i.e., protein exclusively identified in either scLRP1+/+ or scLRP1-/-) was only considered relevant if all scLRP1+/+ samples or all scLRP1-/- samples expressed the protein. The missing values were imputed with the minimum intensity value for each specific data set; (b) for samples expressed in both scLRP1+/+ and scLRP1-/- tissue, the filtering process required 2 or more proteins to be detected in both scLRP1+/+ and scLRP1-/- samples. False discovery analysis was performed using the Benjamini, Krieger, and Yekutieli method using GraphPad Prism 10.0 software. Causal analysis of proteomic data was performed in IPA upstream analysis software (QIAGEN). For IPA, the binary values were imputed using local minimum intensities. Enrichment analyses for gene ontology (biological process) were performed using clusterProfiler 4.2.2 R package on R 4.1.0. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD038236. Global quantification of protein expression. Sciatic nerves from scLRP1+/+ and scLRP1-/- mice were rinsed in PBS to remove the blood and frozen with liquid nitrogen in cryogenic storage tubes (#5016-0001, Thermo Fisher Scientific). Fractured tissue was transferred to a 1 mL milliTUBE containing an AFA fiber (#520135, Covaris catalog) in 200 μL of 50 mM HEPES pH 8.5, 150 mM NaCl, and 2% Triton X-114 and sonicated with a M220 Focused-Ultrasonicator (#500295, Covaris catalog). Sonication parameters were temperature 15 °C, peak power 75 W, duty factor 26, cycles/burst = 1,000, and duration 600 seconds. Extracted proteins were clarified of insoluble material by centrifugation at 15,000 x g for 20 minutes at 4 °C. Protein concentrations were determined with the Micro BCA colorimetric assay (Pierce,...I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
