Computational protocol: Proteomic analysis of lung metastases in a murine breast cancer model reveals divergent influence of CTSB and CTSL overexpression

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Protocol publication

[…] Intact metastases were lysed with 4 % SDS in PBS and boiled at 95 °C for 30 min, followed by ultrasonication. Cell debris was removed by centrifugation and proteins were precipitated by addition of 9 volumes of acetone and 1 volume of methanol. For each genotype, the proteomes of three independent biological replicates were measured by LC‑MS/MS. When necessary, precipitated samples of several mice were pooled prior to labeling, thus enabling a pre-fractionation step prior to LC‑MS/MS. Supplementary Table gives an overview about the pooled samples. Between 3 and 10 metastases per mouse were used for LC-MS/MS sample pools. Due to differences in initial availability, tgCTSL metastasis lysates were pre-fractionated on a HPLC SCX column, while tgCTSB lysates were pre-fractionated using a STAGE‑TIP SCX protocol .Trypsin digestion and differential dimethylation were essentially performed as described previously . LC-MS/MS was performed as described previously using an Orbitrap XL mass spectrometer (Thermo Scientific) . Peptide spectrum matching and relative protein quantitation were performed as described previously , with the following changes: the validated (Swiss-Prot) UniProt mouse proteome was downloaded on November, 17th, 2015. Sequences of human CTSB and CTSL were added manually. The GPM contaminant database with additional common mycoplasma contaminants were appended. The database was appended with an equal number of randomized sequences derived from the original mouse proteome entries. An FDR of < 1 % was used both at peptide and at protein ID level. Proteins with only one identified peptide were only accepted when measured in all three replicates of one genotype. The relative quantitation was performed on MS1 level using the XPRESS algorithm . Protein fold change (FC) values were calculated as log2 (abundance tgCTS / abundance WT). The FC values were normalized to center at zero. Statistical significance was tested using the linear model for microarray data (limma) as described previously . Raw data and all relevant data generated during analysis, including the used databases have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD005860. [...] The subcellular localization of proteins was determined using ngLoc . In order to identify clusters of co-regulated, functionally related proteins, we performed a gene ontology (GO) enrichment analysis, with a focus on the “cellular compartment” and “biological process” annotation . We chose the TopGO algorithm to minimize GO term redundancy . Only clusters with a p-value < 0.05 and including at least three significantly affected proteins were considered. Hierarchical clustering was performed using the Multi Experiment Viewer (version 4.9.0) software with standard settings. […]

Pipeline specifications

Software tools XPRESS, limma, ngLOC, TopGO
Databases ProteomeXchange
Applications MS-based untargeted proteomics, Protein sequence analysis
Organisms Mouse mammary tumor virus, Mus musculus, Homo sapiens
Diseases Breast Neoplasms, Neoplasms, Machado-Joseph Disease
Chemicals Cysteine