Computational protocol: Establishment of a tagged variant of Lgr4 receptor suitable for functional and expression studies in the mouse

Similar protocols

Protocol publication

[…] The samples were cleaved directly on beads with 1 µg of trypsin (Sigma-Aldrich) at 37 °C overnight (Masuda et al. ). Neutral lost scan mass spectrometry (NLS-MS) analysis was performed according to Hebert et al. (). All data were analyzed and quantified with MaxQuant software (version 1.5.3.8) (Cox et al. ). The false discovery rate (FDR) was set to 1% for both proteins and peptides, and we specified a minimum length of seven amino acids. The Andromeda search engine was used for the MS/MS spectra search against the Mus musculus database (downloaded from Uniprot in March 2015, containing 44,654 entries). Enzyme specificity was set as C-terminal to Arg and Lys, also allowing cleavage at proline bonds and a maximum of two missed cleavages. Dithiomethylation of cysteine was selected as fixed modification and N-terminal protein acetylation and methionine oxidation as variable modifications. The “match between runs” feature of MaxQuant was used to transfer identifications to other LC–MS/MS runs based on their masses and retention time (maximum deviation 0.7 min) and this was also used in quantification experiments. Quantifications were performed with the label-free algorithms. Data analysis was performed using Perseus 1.5.2.4 software (Tyanova et al. ). […]

Pipeline specifications

Software tools MaxQuant, Andromeda, Perseus
Application MS-based untargeted proteomics
Organisms Mus musculus