Computational protocol: The PHD finger protein Spp1 has distinct functions in the Set1 and the meiotic DSB formation complexes

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Protocol publication

[…] Protein preparation for mass spectrometry-based proteomic analyses was as described []. Briefly, extracted proteins were stacked in the top of a SDS-PAGE gel (NuPAGE 4–12%, Invitrogen) before in-gel digestion using trypsin (Promega, sequencing grade). Resulting peptides were analysed by online nanoLC-MS/MS (UltiMate 3000 coupled to LTQ-Orbitrap Velos Pro and Ultimate 3000 RSLCnano coupled to Q-Exactive Plus, Thermo Scientific, for Spp1 and Mer2 interactomes analyses, respectively) using a 120-min gradient. Peptides and proteins were identified and quantified using MaxQuant (version 1.5.8.3 []) and SwissProt database (June 2017 version, Saccharomyces cerevisiae S288c taxonomy). Only proteins identified with a minimum of two unique + razor peptides were taken into account for further analyses. Statistical analyses were performed using ProStaR []. Proteins identified in the reverse and contaminant databases, proteins only identified by site and proteins exhibiting less than 3 intensity values in one condition were discarded from the list. After log2 transformation, intensity values were normalized by median centering before missing value imputation (replacing missing values by the 2.5 percentile value of each column); statistical testing was conducted using limma t-test. Differentially recovered proteins were sorted out using a log2(fold change) cut-off of 7 and a FDR threshold on remaining p-values of 1% using the Benjamini-Hochberg method. […]

Pipeline specifications

Software tools MaxQuant, ProStaR, limma
Application MS-based untargeted proteomics
Organisms Saccharomyces cerevisiae