Computational protocol: Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix

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Protocol publication

[…] Protein identification and relative quantification was carried out with the software MaxQuant [] (version 1.2.0.18, Max Planck Institute of Biochemistry, Munich, Germany). Peptides with the same sequence but different labeling states elute at the same retention time. Heavy to light peptide pairs can be detected by their distinct mass shifts according to the labeling with heavy arginine and lysine. MaxQuant uses the intensity of heavy and light labeled peptide pairs to calculate relative peptide abundances. The derived peptide intensity ratios belonging to the same protein are the basis for relative protein quantification.Within the MaxQuant workflow, database searching was carried out by the Andromeda search engine [] against a reverse concatenated IPI human database (version 3.68) including a contaminant list. Recalibration of precursor masses by the option “first search” with a 20 ppm mass tolerance against the human first search database provided by MaxQuant.org. Trypsin with maximum two missed cleavages was set as protease. Carbamidomethylation of cysteine was specified as fixed modification, and oxidation of methionine and acetylation of the protein N-terminal were defined as variable modifications. A peptide mass tolerance of 6 ppm was applied. For tandem MS identification six top peaks per 100 Da were chosen and searched with a fragment ion mass tolerance of 0.5 Da.Peptide and protein false discovery rates were limited to 1 %. Protein identification required at least two unique peptides. The minimal peptide length was set to six amino acids. For protein quantification, the minimal peptide ratio count was set to 2. The option “match between runs” was used for samples measured within the same batch. Re-quantification of proteins was also applied.Proteins with a log2 fold change (FC) above 0.5 or below −0.5 were considered to be up- respectively down-regulated. Furthermore, only proteins showing in at least three out of four replicates regulation in the same direction and an average FC of all replicates fulfilling the criteria for regulation were considered as significantly regulated.For identification of significantly regulated clusters of functionally related regulated proteins the web-based bioinformatics tool DAVID [] was used. The list of regulated proteins was subjected to DAVID, whereas all identified proteins served as background for cluster analysis. Protein clustering was performed according to biological and molecular function derived from the PANTHER classification system []. […]

Pipeline specifications

Software tools MaxQuant, Andromeda, DAVID
Application MS-based untargeted proteomics
Organisms Homo sapiens