Proteome discoverer software v1

All MSMS data generated was searched against a customised database for Wolbachia (wBm) and B. malayi (concatenated from .fasta files obtained from UniProtKB www.uniprot.org, downloaded on 14/01/2013) using the search engine MASCOT and Proteome Discoverer software v1.2 (Thermo Scientific, UK). Search parameters included a precursor mass tolerance of 10 ppm and fragment ion tolerance 0.8 Da with one tryptic missed cleavage permitted. Carbamidomethyl (C) was set as a static modification with oxidation of methionine (M), deamidation (N, Q) and phospho (ST) (Y) set as dynamic modifications. A decoy database was searched and relaxed peptide confidence filters applied to the dataset (ion scores p < 0.05/FDR 5%).

Tryptic digests were analyzed by LC-MS/MS as previously described (Jorissen et al., 2017 (link)), using an easy-nanoLC 1000 liquid chromatograph (Thermo Fisher Scientific) on-line coupled to a LTQ-Orbitrap Velos Pro (Thermo Fisher Scientific). Analysis of the mass spectrometric raw data was carried out using Proteome Discoverer software v.1.2 (Thermo Fisher Scientific) with built-in Sequest v.1.3 and interfaced with an in-house Mascot v.2.4 server (Matrix Science). MS/MS spectra were searched against dd_Djap_V1.contigs.orf.fasta and smedV4.contigs.orf_1.fasta databases (kindly provided by Jochen Rink, Max Planck Institute for Molecular Cell Biology and Genetics, Dresde, Germany). Search engine result files were evaluated in Scaffold v.3.6.1 (Proteome Software, Portland, OR, USA) using the Peptide Prophet and Protein Prophet algorithm, with a preset minimal peptide and protein identification probability of 95% and 99%, respectively.

The raw data files were processed and quantified using Proteome Discoverer software v1.4 (Thermo Fisher Scientific) and searched against the UniProt Human database (downloaded 29/06/17; 140,000 entries) plus the ASFV I215 protein sequence using the SEQUEST algorithm. Peptide precursor mass tolerance was set at 10 ppm, and MS/MS tolerance was set at 0.6 Da. Search criteria included carbamidomethylation of cysteine (+ 57.0214 Da) as a fixed modification and oxidation of methionine (+ 15.9949 Da) and SILAC labels [+ 6.02 Da (R) or + 10.008 Da (R) and + 4.025 Da (K) or + 8.014 Da (K)] as variable modifications. Searches were performed with full tryptic digestion and a maximum of 1 missed cleavage was allowed. The reverse database search option was enabled and all peptide data were filtered to satisfy a 1% false discovery rate (FDR). Contaminants, reverse database hits and hits corresponding to one single peptide were removed. Protein ratios were calculated and converted into their log2. Putative interaction partners were selected when their ratios were above the cut-off (mean + 1.96 SD) and had been identified in at least two of the three replicates unless otherwise indicated. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD023086.

Protein spots with ≥2 fold differential regulation were analysed using nano reverse phase-LC (Thermo Scientific, USA) coupled with an Orbitrap Elite Mass spectrometer (Thermo Scientific, USA) using standard parameters^{71 (link)}. The MS data acquisition was done in positive ion mode (m/z 350–4000 Da) using Xcalibur software (Version 2.2.SP1.48, Thermo Scientific, USA). Protein identification was done with Proteome Discoverer software v.1.4 (Thermo Scientific) using the following parameters: 2 missed cleavages, 10 ppm and 0.5 Da as precursor mass tolerance and fragment mass tolerance, respectively. In addition, cystine carbamidomethylation as fixed modification, methionine oxidation, N-terminal acetylation and phosphorylation (S, T, Y) as variable modification.
Protein spots with 1.5 to 1.9 fold differential regulation were analysed using MALDI-TOF/TOF analysis. Prior to analysis, internal calibration was carried out using TOF-Mix™ (LaserBio Labs, France). For MALDI-TOF/TOF analysis, 1 µL of purified peptides were mixed with 1 µL of the alpha-cyano-4-hydroxy cinnamic acid matrix (10 mg/mL) on MALDI target plate. Mass spectra were acquired in positive reflectron mode and the monoisotopic peak list (m/z range of 700–4000 Da) was used for protein identification using MS-Fit (http://prospector.ucsf.edu) online software using standard parameters^{69 (link)}.

CiGEnCs stably expressing GFP-VEGFR2 or GFP were serum-starved for 1 h and then stimulated with 40 ng/ml VEGF for the indicated time points. The cells were then washed once with ice-cold PBS and harvested in lysis buffer (20 mM Tris pH7.5, 137 mM NaCl, 0.5% NP-40, 1 mM EDTA, 2 mM sodium orthovanadate, 10 mM sodium fluoride) containing protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were clarified by centrifugation for 12 min at 12,000 × g and 4 °C. The lysates were then incubated with GFP-Trap agarose beads (ChromoTek, Martinsried, Germany) for 1 h with end-over-end rotation at 4 °C. The beads were washed three times with lysis buffer and the bound protein eluted by heating at 95 °C with SDS-PAGE sample buffer. Samples were run approximately 1 cm into the separating gel of an SDS-PAGE gel and then subjected to in-gel tryptic digestion. The resulting peptides were fractionated using an Ultimate 3000 nanoHPLC system in line with an LTQ-Orbitrap Velos mass spectrometer (ThermoFisher Scientific). The raw data files were processed and quantified using Proteome Discoverer software v1.4 (ThermoFisher Scientific) and searched against the UniProt Human database using the SEQUEST algorithm.

The raw data files were processed and quantified using Proteome Discoverer software v1.4 (Thermo Scientific) and searched against a combined database consisting of the UniProt human database (downloaded 8 November 2014 : 126 385 entries) and adenovirus protein sequences using the SEQUEST algorithm. Peptide precursor mass tolerance was set at 10 ppm and MS/MS tolerance was set at 0.6 Da. Search criteria included oxidation of methionine (+15.9949) as a variable modification and carbamidomethylation of cysteine (+57.0214) and the addition of the TMT (+229.163) to peptide N-termini and lysine as fixed modifications. Searches were performed with full tryptic digestion and a maximum of one missed cleavage was allowed. The reverse database search option was enabled and all peptide data were filtered to satisfy a false discovery rate (FDR) of 1 %.