Computational protocol: Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome

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Protocol publication

[…] A transcript encoding the A. rubens luqin-type precursor (ArLQP) has been identified previously (GenBank: KT601719;). Here a cDNA containing the complete open reading frame of ArLQP was amplified by PCR from A. rubens radial nerve cord total cDNA using specific primers (see supplementary Table ) and Q5 proofreading polymerase (NEB; Cat. No. M0491S), cloned into pCR-Blunt II TOPO vector (Invitrogen; Cat. No. K280002) and sequenced (TubeSeq service; Eurofins Genomics). The amino acid sequence of ArLQP was aligned with luqin/RYamide-type precursors from other species (see supplementary Table  for a list of the sequences) using MAFFT version 7 (5 iterations, substitution matrix; BLOSUM62) and highlighted using the software BOXSHADE (www.ch.embnet.org/software/BOX_form.html) with 70% conservation as the minimum for highlighting. [...] Radial nerve cords from two specimens of A. rubens were dissected and transferred to a micro-centrifuge tube containing 3% acetic acid (in ddH2O). The tube was incubated in a boiling water bath for 10 minutes. The nerve cords were then sonicated and homogenized to lyse cells. The extract was centrifuged, supernatant transferred to a glass vial and solvent was bubbled-off using nitrogen gas. Frozen radial nerve cord extracts were thawed and an aliquot diluted 10-fold with 0.1% aqueous formic acid, then filtered through a 0.22 μm Costar Spin-X centrifuge tube filter to remove particulates. The extract was analysed by means of nanoflow liquid chromatography with electrospray ionisation quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-MS/MS) using a nanoAcquity UPLC® system coupled to a Synapt G2 HDMS mass spectrometer and MassLynx v4.1 SCN 908 software (Waters Corporation, Milford, MA, USA). The mobile phases used for the chromatographic separation were: 0.1% aqueous formic acid (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). An aliquot containing 15 μL of the extract was applied to a trapping column (Symmetry C18 180 μm × 20 mm, 5 μm particle size, 100 Å pore size, Waters Corporation) using 99.9% mobile phase A at a flow rate of 10 μL min−1 for 3 min, after which the fluidic flow path included the analytical capillary column (HSS T3 75 μm × 150 mm, 1.8 μm particle size, 100 Å pore size, Waters Corporation). A linear gradient of 5–40% mobile phase B over 105 min was utilized with a total run time of 120 min. Nanoflow ESI source conditions were as follows: capillary voltage 3.5 kV, sample cone voltage 25 V with a source temperature of 80 °C. The instrument was operated in resolution mode (~20,000 measured at full width half height). A data-dependent acquisition was performed that would trigger an MS/MS scan on any multiply charged peptide of intensity ≥ 450 counts/sec within the survey scan m/z range 300–1950. A maximum of 5 precursor peptides were selected for MS/MS from each survey scan and MS/MS data collected for 6 scans then combined. Each peptide precursor was then excluded from selection for MS/MS for a period of 20 sec. MS/MS data were collected over m/z range 50–1950 using m/z and charge state dependent collision energy applied to the trap region. Tandem mass spectra were extracted by ProteinLynx Global Server version 2.5.1 (Waters Corporation, Milford, MA, USA) with charge state deconvolution and deisotoping performed prior to creation of a peak list file for each sample. A peak list file generated from acetic acid extract data was used to interrogate protein database UniProtKB/TrEMBL release 2018_02 filtered for taxon identifier 7586 (phylum Echinodermata) containing 70,885 sequences and 29,368,428 residues (http://www.uniprot.org/). Search parameters used by Mascot software (Matrix Science, London, UK; version 2.5.1) were “none” for enzyme i.e. Mascot searched each protein sequence for every sub-sequence meeting the remaining search criteria, precursor mass error less than 5 ppm and fragment ion tolerance 20 mDa. A variable modification of C-terminal amidation was permitted. […]

Pipeline specifications

Software tools MAFFT, BoxShade, PLGS
Applications MS-based untargeted proteomics, Proteome data visualization
Chemicals Tachykinins