Geoduck clams are an increasingly important aquaculture product in the Pacific Northwest of the United States, while also holding essential ecological roles in the ecosystem. These long-lived clams are very fecund, but hatchery production of larvae is hindered by the inability to sex a live geoduck, thus resulting in asynchronous spawning of broodstock and unequal sex ratios. During geoduck reproductive maturation, the physiology of the gonad changes from sexually undifferentiated connective tissue to sex-specific, mature reproductive cells that can be released into the water column. We applied proteomics tools to uncover the cellular mechanisms underlying these physiological changes in the gonad. Sex- and stage-specific proteins were characterized using data dependent acquisition (DDA), or whole proteome profiling, on gonad tissue. Gonad proteomes became increasingly divergent between males and females as maturation progressed. The DDA data were leveraged to develop biomarkers of geoduck sex and maturation stage, analyzed with selected reaction monitoring (SRM) in gonad and hemolymph. The SRM assay yielded a reduced suite of peptides that can be used as an efficient assay to non-lethally determine geoduck sex and maturation stage pre-spawning. This is one of a few examples of cutting-edge proteomics being used to develop applicable tools for the aquaculture industry.

A subset of proteins were chosen for development of a suite of targeted assays using selected reaction monitoring (SRM) on the mass spectrometer (MS). Based on the data-dependent acquisition, proteins that were detected only in one of these stages; early stage males (EM), early stage females (EF), late stage males (LM), or late stage females (LF) were screened for usable peptide transitions in SRM in Skyline in gonad tissue. Data independent acquisition (DIA) was used to generate spectral libraries for biomarker development in the gonad tissue (Figure 1). Equal amounts of isolated peptides from the three biological replicates for EM, EF, LM, and LF used in the DDA experiment (described above) were pooled in equal quantities for DIA on the Q-Exactive HF (Thermo). Each sample included a spiked-in internal quality control peptide standard (375 fmol PRTC + BSA; Pierce, hereafter referred to as “QC”). Sample injections for all DIA experiments included 1 ug protein plus the QC in a 2 µl injection. An analytical column (27cm) packed with 3 µm C18 beads (Dr. Maisch) and a 3 cm trap with 3 µm C12 beads (Dr. Maisch) were used for chromatography. Technical replicate DIA spectra were collected in 4 m/z isolation width windows spanning 125 m/z ranges each (400-525, 525-650, 650-775, 775-900; Panchaud et al., 2009). For each method, a gradient of 5-80% ACN over 90 minutes was applied for peptide spectra acquisition. Raw data can be accessed via ProteomeXchange (http://www.proteomexchange.org/) under identifier PXD004921. MSConvert (Chambers et al., 2012) was used to generate mzML files from the raw DIA files. In order to generate spectral libraries for targeted method development, Peptide Centric Analysis was completed with the software program PECAN (Ting et al., in review; Ting et al., 2015). Input files included the list of peptides generated for SRM (n=212), as described above, and the mzML files generated from the raw DIA files. PECAN correlates a list of peptide sequences with the acquired DIA spectra in order to locate the peptide-specific spectra within the acquired DIA dataset. A background proteome of the in silico digested geoduck gonad proteome was used. The PECAN .blib output file was then imported into Skyline daily v. 3.5.1.9706 (MacLean et al., 2010) to select peptide transitions and create MS methods that would target specific peptides and transitions. Peptide transitions are the reproducible fragments of peptides that are generated during the MS2 scan in a MS. Peptides reliably fragment in the same way in the MS, therefore transitions are a robust and consistent signal of a peptide’s presence (Wolf-Yadlin et al., 2007). Peptide transitions were selected if peak morphology was uniform and consistent across the MS2 scans for technical replicates. Peptides were selected for targeted analysis if they had > 3 good quality transitions and there were > 2 peptides per protein. A maximum of 4 transitions per peptide were selected for targeted analysis and no more than 3 peptides per protein were selected. The final list included 25 transitions for EM biomarkers, 22 for EF, 133 for LM, and 52 for LF. This transition list was divided between two method files for the final SRM analyses to provide adequate dwell time on individual transitions in order to accurately detect and measure all peptides desired (Picotti and Aebersold, 2012). Selected reaction monitoring (SRM) was carried out on a Thermo Vantage for all eighteen geoduck gonad samples used in the original DDA analysis. Samples were prepared as described above for DIA (1 µg of protein per 3 µl injection). A new C18 trap (2 cm) and C18 analytical column (27.5 cm) were used and each sample was analyzed in triplicate across two MS experiments to cover the entire peptide transition list (n=212). Raw data can be accessed in the PeptideAtlas (http://www.peptideatlas.org/PASS/PASS00943) under accession PASS00943. In addition to the peptides selected from the gonad proteome, transitions from five proteins were added to the assay for hemolymph SRM analysis. These proteins were selected based on gonad proteome annotation to include 1) proteins that would likely be circulating in the hemolymph and 2) gonad proteins that had homology with the mussel hemolymph proteome. The proteins are vitellogenin (2 proteins), glycogen synthase (2 proteins), and glycogenin-1. PECAN was run as described above to use Skyline to select hemolymph protein transitions. For the new hemolymph proteins, the minimums for peptides and transitions could not be met for every protein but were still included in the analysis (n=40 transitions). These transitions, and the previous gonad transitions, were analyzed across the eighteen geoduck hemolymph samples in two technical replicates, as described above for the gonad. Some of these peptides yielded no data in the hemolymph, resulting in a dataset of 171 peptide transitions. Raw data can be accessed in PeptideAtlas (http://www.peptideatlas.org/PASS/PASS00942) under accession PASS00942.

Geoduck gonad tissue and hemolymph from 18 clams: males and females from early, middle, and late maturation stages.