Mascot v2

AP-MS experiments followed a GeLC MS/MS approach. Gel lanes were fragmented into eight equally sized pieces and subjected to in-gel trypsin digestion using a Perkin Elmer Janus Automated Workstation. Peptide mixtures were acidified to 0.1% TFA and injected onto a nanoACQUITY UPLC (Waters Corporation) coupled to an LTQ-Orbitap XL (Thermo Fisher Scientific) via an Advion Biosciences Nanomate. Peptides were eluted over a 30 min gradient (5–40% ACN). Mascot distiller was used to extract peak lists, which were searched with Mascot v.2.4.1 (Matrix Science; RRID:SCR_014322) against the Drosophila melanogaster Uniprot reference proteome. Methionine oxidation was entered as a variable modification and search tolerances were 5 ppm and 0.8 Da for peptides and fragments, respectively. Individual lane searches were combined and results compiled in Scaffold 4.0.3 (RRID:SCR_014345).

The raw mass spectra were deconvoluted using Proteome Discoverer v1.4.1.14 (Thermo Fisher Scientific, Waltham, MA). The spectra were searched against Mus Musculus (Swissprot, January 2015) supplemented with human Matrin-3 using Mascot v2.4.1 (Matrix Science, London, UK) with the following variable modifications: oxidation (Met) and carbamidomethyl (Cys). Mass tolerances for precursor ions were set at ± 10 ppm, for fragment ions at ± 0.8 Da. A maximum of 2 missed cleavages was allowed. Data were processed for label-free quantitation using Scaffold v4.5.1 (Proteome Software Inc., Portland, OR) and X!Tandem (The GPM, v2010.12.01.1) to further improve confidence in protein identification. At least 2 peptides were required for protein identification, with 0.1% peptide FDR and 1% protein FDR. Only exclusive spectral counts were used for prediction of protein-protein interactions.

All MS/MS samples were analysed using Mascot v2.4.1 (Matrix Science). Mascot was set up to search a database comprised of the gene models identified within the F. hepatica genome (version 1.0, 101,780 entries; accession PRJEB6687 [14 ]) assuming trypsin digestion with 1 missed cleavage permitted. Mascot was searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 10.0 ppm. Carbamidomethylation of cysteine was specified in Mascot as a fixed modification. Gln->pyro-Glu of the N-terminus, oxidation of methionine, dioxidation of methionine, acetyl of the N-terminus, BHAc of lysine and NHS-LC-Biotin of the N-terminus were specified in Mascot as variable modifications. An additional search against the NCBI database or Ovis aries gene models (http://www.ensembl.org/Ovis_aries/Info/Index) was run to identify potential host proteins.

Proteins in the various EV fractions were reduced with 2 mm DTT in 50 mm NH₄HCO₃ (60 °C/20 min) and alkylated with 5 mm iodoacetamide (room temperature (18–21 °C) in the dark/30 min). Samples were then incubated with 100 ng/μl sequencing grade trypsin (Promega) (37 °C/overnight). The digestions were stopped by the addition of trifluoroacetic acid (TFA) to a final concentration of 0.1% and dried in a vacuum centrifuge. The final mixtures were reconstituted with 10 μl of 0.1% TFA before analysis by LC-MS/MS. Five microliters of the resulting suspension were delivered to an analytical column (Eksigen C18-CL NanoLC Column, 3 μm; 75 μm × 15 cm) equilibrated in 5% acetonitrile/0.1% formic acid (FA). Elution was carried out with a linear gradient of 5–35% buffer B in buffer A for 30min (buffer A: 0.1% FA; buffer B: acetonitrile, 0.1% FA) at a flow rate of 300 nl/min. Peptides were analyzed in a nanoESI qQTOF mass spectrometer (5600 TripleTOF, ABSCIEX) operating in information-dependent acquisition mode, in which a 0.25-s TOF MS scan from 350–1250 m/z, was performed, followed by 0.05-s product ion scans from 100–1500 m/z on the 50 most intense 2–5 charged ions. Peak list files were generated by Protein Pilot v4.5 (Applied Biosystems) using default parameters and exported to Mascot v2.4.1 (Matrix Science) for database searching.