| Metadata |
| datasetIdentifier | PASS00942 |
| datasetType | SRM |
| submitter | Emma Timmins-Schiffman <emmats@uw.edu> |
| submitter_organization | University of Washington |
| lab_head_full_name | Steven Roberts |
| lab_head_email | sr320@uw.edu |
| lab_head_organization | University of Washington |
| lab_head_country | USA |
| datasetTag | geoduckhemolymph |
| datasetTitle | Selected reaction monitoring of geoduck hemolymph peptides to develop biomarkers of reproductive maturation status |
| publicReleaseDate | 2016-10-20 00:00:00 |
| finalizedDate | 2016-10-20 13:44:11 |
| summary | Geoduck clams are an increasingly important aquaculture product in the Pacific Northwest of the United States, whilealso 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 mantle 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. |
| contributors | Emma Timmins-Schiffman, Grace Crandall, Brent Vadopalas, Michael Riffle, Steven B. Roberts |
| publication | unpublished |
| growth | Geoduck clams were collected in November 2014 from Nisqually Reach, Washington, USA. Clams were collected at depths between 9 to 14 meters from a sandy substrate. Geoduck were housed at Taylor Shellfish for the duration of their reproductive maturation and regularly sampled for histology, and proteomics of gonad and hemolymph. |
| treatment | |
| extraction | |
| separation | |
| digestion | Hemolymph protein content was determined using Pierce's BCA assay and a volume containing 50 ug of protein for each sample was evaporated to 20 ul and then resuspended in 100 ul of 6M urea in 50 mM NH4HCO3 for sonication. Each sample was incubated with TCEP BUFFERED AT pH 8.8 (1 hr, 37C). Samples were alkylated with iodoacetamide (IAM; 1 hr, 20C) followed by a 1 hour incubation with dithiothreitol to absorb any remaining IAM. To each sample, NH4HCO3 and HPLC grade methanol were added to dilute urea and to increase solubilization of membrane proteins. Samples were digested overnight with trypsin at 37C. Digested samples were evaporated an reconstituted in 5% acetonitrile (ACN) + 0.1% trifluoroacetic acid (TFA) (100 ul) and pH was decreased to <2. Desalting of the samples was done using Macrospin columns (The Nest Group) following the manufacturer's protocol. Dried peptides were reconstituted in 100 ul of 5% ACN + 0.1% formic acid. |
| acquisition | SRM was carried out on a Thermo Vantage. Each sample included a spiked-in internal quality control peptide standard (375 fmol PRTC + BSA; Pierce). One ug of protein was injected in 3 ul. A C18 trap (2 cm) and C18 analytical column (27.5 cm) were used. All samples were analyzed in duplicate across two MS experiments to cover the full list of peptide transitions. |
| informatics | Acquired SRM data were analyzed in Skyline for peptide transition quantification. |
| instruments | Thermo TSQ Vantage |
| species | Panopea generosa |
| massModifications | none |
