Computational protocol: Label-free LC-MS analysis of HER2+ breast cancer cell line response to HER2 inhibitor treatment

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Protocol publication

[…] Label-free LC-MS analysis was carried out using Progenesis QI for proteomics software version 4.1 (NonLinear Dynamics, UK), as recommended by the manufacturer (see www.nonlinear.com for further background to alignment, normalisation, calculation of peptide abundance, etc.). As already described by Meleady et al. [] the software processed the raw data in two steps. Firstly each sample run was subjected to alignment which involved aligning the data based on the LC retention time of each sample; this allows for any drift in retention time giving an adjusted retention time for all runs in the analysis. The sample run that yielded most features (i.e. peptide ions) was used as the reference run, to which retention time of all of the other runs were aligned and peak intensities were normalised. The Progenesis peptide quantification algorithm calculates peptide abundance as the sum of the peak areas within its isotope boundaries. Each abundance value is then transformed to a normalised abundance value by applying a global scaling factor. Protein abundance was calculated as the sum of the abundances of all peptide ions which have been identified as coming from the same protein. A number of criteria were used to filter the data before exporting the MS/MS output files to MASCOT (www.matrixscience.com) for protein identification; peptide features with ANOVA (analysis of variance) p-value ≤0.05 between experimental groups, mass peaks (features) with charge states from +1 to +3, and greater than 3 isotopes per peptide. All MS/MS spectra were exported from Progenesis software as a MASCOT generic file (mgf) and used for peptide identification with MASCOT (version 2.3) searched against the UniProtKB–SwissProt database (taxonomy, homo sapiens). The search parameters used were as follows: peptide mass tolerance set to 20 ppm, MS/MS mass tolerance set at 0.6 Da; up to two missed cleavages were allowed, carbamidomethylation set as a fixed modification and methionine oxidation set as a variable modification. Only peptides with ion scores of 40 and above were considered and re-imported back into Progenesis QI for proteomics software for further analysis. A number of criteria were applied to assign a protein as identified; proteins with ≥2 peptides matched, a ≥1.5 fold difference in abundance and an ANOVA between experimental groups of ≤0.05.The biological function of the proteins identified was assigned using ontology tools in PANTHER []. […]

Pipeline specifications

Software tools Progenesis QI, PANTHER
Applications MS-based untargeted proteomics, Protein sequence analysis
Organisms Homo sapiens
Diseases Breast Neoplasms, Neoplasms, Drug-Related Side Effects and Adverse Reactions
Chemicals Tyrosine