Cilia and flagella serve as cellular antennae that translate sensory information into cellular responses. In sperm, a single chemoattractant molecule triggers signaling events that evoke a Ca2+ response and chemotactic steering. Key components of the chemotactic signaling pathway from sperm of the sea urchin Arbacia punctulata were investigated. Targeted proteomics employed a spiked-in heavy isotope-labelled standard protein (QConCat) and in-gel protein digestion with trypsin. The standard protein together with the previously determined copy number of the receptor guanylate cyclase allowed determining the copy numbers for 19 polypeptides of this signaling pathway.

The collection of A. punctulata sperm and the preparation of flagella are described in Trötschel et al. (EMBO J. 2019). An aliquot of flagella from six animals was resuspended and incubated for 30 minutes on ice in lysis buffer (10 mM Tris/HCl, pH 7.6; 140 mM NaCl, 1 mM EDTA, 1% dodecyl-maltopyranoside (DDM), protease inhibitor cocktail 1:500 (P8340, Sigma-Aldrich, USA). After centrifugation at 10,000 x g and 4°C, the supernatant was stored in a new tube, and the protein content was determined by a BCA assay. Sperm flagella samples were mixed with known amounts of the standard protein and were subjected to 12.5% (v/v) SDS-PAGE. SDS-PAGE was stopped after the sample migrated about 1 cm into the separation gel, and the gel was stained with Coomassie Blue. The ca. 1 cm long gel piece was cut into 1-2 mm-sized pieces. In-gel digestion was done as described (Pichlo et al., 2014) with 6.25 ng/µl trypsin in a NH4HCO3 buffer (40 mM at pH 8.6). The LC-MS/MS system consisted of a TSQ Vantage triple quad MS (Thermo Fisher Scientific, USA) interfaced with a nanoAcquity UHPLC-system (Waters, USA). LC used an Acquity UPLC M-Class Symmetry C18 trap column (5 µm particle size, 180 µm x 20 mm, 2D, V/M) and an Acquity UPLC M-Class HSS T3 analytical column (1.8 µm particle size, 75 µm x 150 mm) (Waters, USA). LC conditions (66-min multistep gradient from 1% to 99% acetonitrile/0.1% formic acid at a flow rate of 0.4 µl min-1) were as described (Pichlo et al., 2014). SRM parameters for the TSQ Vantage and data analysis with Skyline 3.7 were essentially as described (Pichlo et al., 2014) with the following changes: the two most intense and specific fragment ions (transitions) were used for quantification, a measuring time window of 2.5 min from each peak apex as well as 3-s cycle time were set. For optimal ion transmission, the following S-lens RF amplitude values were fixed: 53 at 182.082 m/z, 109 at 508.208 m/z, and 220 at 997.398 m/z. The collision energy was first predicted by Skyline 3.7 and then individually optimized for each precursor ion.

For each protein, 2 to 4 tryptic peptides were selected based on previous shotgun datasets as well as prediction and validation from targeted proteomics. A synthetic protein containing the selected peptides and heavy isotope (15N, 13C)-labeled lysine and arginine residues was used as internal standard. For quantification, an aliquot of 20 µg sperm flagella protein was spiked with either 0.15, 1.5, or 15 pmol heavy standard protein to account for different abundance of sperm target proteins. Six different animals were analysed in two experimental sessions. For samples of session 1, animals were indicated as #1, #6, and #8. For samples of session 2, animals were indicated as #1, #2, and #6.