Orbitrap xl mass spectrometer

In-gel digestion and LC–MS/MS analysis were performed in accordance with the work of Fu et al.^{63 (link)}. Sperm basic proteins extracted from sperm nuclei of S. horneri were separated by SDS-PAGE (14% polyacrylamide gel). Gels were stained using CBB staining solution (SP-4010; Integrale, Tokushima, Japan). Four bands around 25 kDa (Fig. 4) were cut using a razor blade. The excised bands were digested with trypsin. The tryptic digests were separated by Paradigm MS2 HPLC (Bruker-Michrom, Auburn, CA, USA) equipped with an HTS-PAL auto-sample injection system (LEAP, Carrboro, NC, USA) on a nanocapillary column (0.1 mm inner diameter × 50 mm; Chemicals Evaluation and Research Institute, Tokyo, Japan). The eluates from the column were subsequently subjected to mass spectral analysis using an Orbitrap XL mass spectrometer (Thermo Scientific, Waltham, MA, USA). MS/MS spectral data were analyzed using Thermo Proteome Discoverer version 1.4.0.208 with the Mascot search engine (Matrix Science, London, UK), using transcriptomic data of S. horneri^{35 (link)}.

Protein identification was performed using nano LC-ESI-MS/MS. The MS system consisted of an Agilent 1100 nanoLC system (Agilent), PicoTip electrospray emitter (New Objective) and an Orbitrap XL mass spectrometer (Thermo-Fisher). Protein spots from the membranes were in-gel digested by trypsin (Promega) (with and without citraconic anhydride treatment) and applied to nanoLC-ESI-MS/MS. Peptides were trapped and desalted on the enrichment column (Zorbax SB C18; 0.3x5 mm; Agilent) for five minutes using 2.5% acetonitrile/0.5% formic acid as eluent, then peptides were separated on a Zorbax 300 SB C18 column (75µmx150mm; Agilent) using an acetonitrile/0.1% formic acid gradient from 5 to 35% acetonitril within 40 minutes. MS/MS spectra were recorded data-dependently by the mass spectrometer, according to manufacturer's recommendations.

Products of ADP-ribosylation reactions containing 10 μg of protein were purified using standard TCA precipitation and resuspended in PBA buffer (100 mM HEPES pH 8.5, 150 mM NaCl, 2 mM MgCl₂). If required, cysteines were reduced in 10 mM dithiotreitol for 30 min at room temperature and alkylated with 10 mM iodoacetamide for 30 min at room temperature in the dark. Proteins were digested with 100 ng trypsin (Promega) overnight at 37 °C and peptides bound to m-aminophenylboronic acid agarose beads (Sigma) for 1 h at 4 °C. The beads were washed extensively with PBA buffer and the ADP-ribosylated peptides eluted with 1 M hydroxylamine (Sigma) pH 7.0 overnight at room temperature. Eluates were purified using C₁₈ ZipTips (Millipore) according to manufacturer instructions and analysed by nano-LC-MS (ThermoFisher U3000 nanoLC and Orbitrap XL mass spectrometer)39 (link). The raw mass spectrometry and tandem mass spectra were converted to.mgf format using Compass39 (link) and searched against the SwissProt database using Mascot (Matrix Science). Search parameters employed a precursor tolerance of 7 p.p.m. and a fragment ion tolerance of 0.8 Da. Quantification of precursor ions employed Skyline40 (link). ADP-ribosylation sites were identified based on a characteristic +15.0109 Da shift on glutamate and aspartate residues41 (link).