Computational protocol: Characterization and use of HapT1-derived homologous tumors as a preclinical model to evaluate therapeutic efficacy of drugs against pancreatic tumor desmoplasia

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Protocol publication

[…] Mass spectrometry data were analyzed by using MaxQuant [, ] software version and searched through an in-house modified UniProt human, mouse, and hamster database, along with the NCBI hamster database by adding common contaminant protein sequences. A database search was performed in Andromeda [] that was integrated in a MaxQuant environment. A list of 280 common laboratory protein contaminants included in MaxQuant was also added to the database, as well as reversed target decoy search, which was used for all sequences. For searching, the enzyme specificity was set to trypsin with the maximum number of missed cleavages set to 2. The precursor mass tolerance was set to 0.07 Da for the first search and then to 0.006 Da for the main search. Mass tolerance for fragment ions in collision-induced dissociation (CID) spectra was set to 0.5 Da. The search included variable modifications of protein N-terminal acetylation, methionine oxidation, and carbamidomethylation of cysteines, and was searched as a fixed modification. The maximal number of modifications per peptide was set to 5. The false discovery rate (FDR) for PSM, proteins, and the site decoy fraction was set to 1%. Additionally, time-of-flight mass spectrometry (TOFMS)/MS mass tolerance was set to be 40 ppm. A minimum peptide length of 6 was set, and the ‘peptide requantification’ function was enabled. To validate and transfer identifications across different runs, the ‘match between runs’ option in MaxQuant was enabled with a retention time window of 0.7 minutes and an alignment time window of 20 minutes. Subsequent bioinformatics analysis were performed using Protein Analysis through Evolutionary Relationships (PANTHER) to compare the gene ontology protein class (GOPC). The obtained PANTHER [] data was further analyzed, and graphs were prepared using Microsoft Excel 2007. The mass spectrometry data has been deposited to the Proteome X- change consortium with the PRIDE partner repository with the database identifier PXD003385 []. […]

Pipeline specifications

Software tools MaxQuant, Andromeda
Application MS-based untargeted proteomics
Organisms Homo sapiens, Mesocricetus auratus
Chemicals Acetylcysteine, Copper, Disulfiram