Computational protocol: Predicting the impact of Lynch syndrome-causing missense mutations from structural calculations

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Protocol publication

[…] The FoldX energy function version 3.1 was used to estimate the free-energy change upon mutations of MSH2 []. FoldX calculations were carried out for each of the monomer structures included in the PDB entry 2O8E [] to assess the reproducibility of the results, and the average is reported here. The RepairPDB function of FoldX was first applied to the wild type structures. Structures for saturation mutagenesis were generated using an in-house Python program that allows for the introduction of all 19 possible point mutants at each position of the protein using multithreading calculations. The BuildModel function of FoldX was employed and five independent runs were carried out and then averaged. The typical prediction error of FoldX is about 0.8 kcal/mol []. [...] For mass spectrometry, stable U2OS cell lines expressing wild type MSH2, L187P and a vector control, were grown to confluency in four 15-cm dishes per cell line. Cells were washed in PBS, and subsequently scraped off the dish in 3 mL of PBS. The cells were harvested by centrifugation and lysed in 1 mL of buffer B for 20 minutes on ice. The lysates were cleared by centrifugation (13000 g, 30 min.), and the supernatants transferred to tubes containing 20 μL TALON metal affinity resin (Clontech). Lysates were incubated for 2 hours at 4°C, after which the beads were washed 4 times in buffer B, followed by addition of 25 μL SDS sample buffer. Samples were prepared in quadruplicates. 17 μL of each sample elution was fractionated by SDS-PAGE (Novex NuPAGE Bis-Tris/MOPS 10% acrylamide) before staining with Coomassie blue, with an estimated protein yield of ~20 μg per lane. Each lane was excised as a single ‘slice’, and tryptic peptides extracted by in gel digestion (0.5μg per slice). Peptide samples were analyzed by LC-MS/MS on a Q Exactive mass spectrometer (Thermo Scientific) coupled to an EASY-nLC 1000 liquid chromatography system (Thermo Scientific) via an EASY-Spray ion source (Thermo Scientific). Peptides were fractionated on a 75 μm x 500 mm EASY-Spray column (Thermo Scientific) over 150 minutes. Precursor ion full scan spectra were acquired over (m/z 300 to 1,800) with a resolution of 70,000 at m/z 400 (target value of 1,000,000 ions, maximum injection time 20 ms). Up to ten data dependent MS2 spectra were acquired with a resolution of 17,500 at m/z 400 (target value of 500,000 ions, maximum injection time 60 ms). Ions with unassigned charge state, and singly or highly (>8) charged ions were rejected. Intensity threshold was set to 2.1 x 104 units. Peptide match was set to preferred, and dynamic exclusion option was enabled (exclusion duration 40 s). Raw MS data files were processed using MaxQuant software (version 1.5.2.8) and searched against UniProtKB human proteome (canonical and isoform sequences). Carbamidomethyl (C) was set as fixed modification and variable modification of acetyl (protein N-term), and oxidized (M) were selected. MaxQuant enzyme specificity was set to trypsin. Lysine and arginine were selected as special amino acid and a maximum number of three missed cleavages were allowed. A minimum peptide length was set to seven residues and a maximum peptide mass was 5,000 Da. A false discovery rate of 1% was set as a threshold at both protein and peptide level, and a mass deviation of 6 parts per million was set for main search and 0.5 Da for MS2 peaks. The match between runs option was selected using the matching and alignment time windows of 0.7 and 20 minutes respectively. Raw intensity values were manually normalized by median ratio of the proteins detected in all samples, and only proteins with four intensities reported in a single set of quadruplicates was carried forward. Zero intensity values were replaced in Persues v 1.5.1.6 (www.biochem.mpg.de/5111810/perseus) from a normal distribution of data based on the input intensities with width and downshift parameters set to default (0.3 and 1.8). Significantly enriched proteins (interactors) were identified by two samples t-test with permutation-based FDR = 0.05 and S0 = 0.2. These are shown as red (103 proteins) among the background of 1483 proteins not satisfying these criteria (see Supporting information ). […]

Pipeline specifications

Software tools FoldX, MaxQuant, Perseus
Databases UniProtKB
Applications MS-based untargeted proteomics, Protein structure analysis
Organisms Homo sapiens
Diseases Colorectal Neoplasms, Hereditary Nonpolyposis, Neoplastic Syndromes, Hereditary, Genetic Diseases, Inborn