Geoduck (Panopea generosa) gonad LC-MS/MS
Geoduck clams (Panopea generosa) were collected from Puget Sound, WA in November, 2014. Male and female geoduck gonads were sampled at three reproductive stages over the course of three months: early, middle, and late. Early stage indicates that the gonad cells are just beginning to differentiate and late stage indicates that the geoduck are ready to spawn. The goal of this study was to identify biomarkers of the geoduck reproductive cycle.
Sample Processing Protocol
Clam gonad tissue was homogenized using a sonicating probe in 300 ul of 6M urea in 50 mM NH4HCO3. The clam tissue was sonicated 3 times and chilled in ethanol with dry ice in between sonications. Protein digestion followed the protocol outlined in Timmins-Schiffman et al. (2014). Briefly, 6.6 ul of 1.5M Tris-HCL (pH 8.8) and 2.5 ul of 200 mM TCEP were added to each sample and then incubated for 1 hour at 37C. Samples were alkylated with 20 ul of 200 mM iadoacetamide and incubated for 1 hour at room temperature (RT) in the dark. Excess IAM was absorbed using 20 ul of 200 mM DTT (1 hour at RT). The remainder of the digestion protocol was carried out on 100 ug of protein from each sample. To each sample, 800 ul of 25 mM NH4HCO3 and 200 ul HPLC grade methanol were added. Trypsin (5 ug) was then added to each sample and incubated overnight at 37C. The digested samples were evaporated to near dryness on a speed vacuum and reconstituted in 100 ul 0.1% formic acid in water and 10 ul 10% formic acid. The samples were evaporated until dry. Before desalting, samples were reconstituted in 100 ul solvent B (5% ACN + 0.1% TFA) and checked to ensure pH < 2. Desalting was done using Macrospin columns (Nest Group). Columns were first prepared with washes of solvents A (60% ACN + 0.1% TFA) and B. Peptides were bound to the column and all contaminants washed off. Peptides were eluted in solvent A, evaporated, and reconstituted in 100 ul solvent B. LC-MS/MS was done on a Q-Exactive-HF (Thermo) on technical triplicates from each sample. The analytical column was 20 cm long and packed with C18 beads with a flow rate of 0.3 ul/min. The solvent gradient was: 0-1 min 98%A/2%B; 1-60 min 95% A; 60-61 min 65% A, 61-71 min 20% A; 71-90 min 98% A.
Data Processing Protocol
A protein identification database was created from translated transcriptomic sequences derived from geoduck gonad tissue. The raw LC-MS/MS data were searched against the database using Comet v 2015.01 rev.2 followed by the Trans-Proteomic Pipeline (downloaded November, 2014).