Geoduck (Panopea generosa) gonad LC-MS/MS DIA
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, 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 hr incubation with bithiothreitol 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 and reconstituted in 5% ACN+0.1% TFA (100 ul) and pH decreased to <2. Desalting of the samples was done using Macrospin columns and dried peptides were reconstituted in 100 ul of 5% ACN + 0.1% formic acid. Equal quantities of peptides were pooled (n=3 per pool) within the groups early female, early male, late female, and late male. LC-MS/MS was accomplished in DIA mode on a Q-Exactive HF. Each sample included a spiked-in internal quality control peptide standard (375 fmol PRTC + BSA). 1 ug of peptide + QC was injected in 2 ul on a 27 cm analytical column with 3 cm trap, both packed with C18 beads. 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, and 775-900 m/z) over a 90 minute gradient of 5-80% ACN.
Data Processing Protocol
mzML files were generated using MSConvert. Peptide Centric Analysis (PECAN) was used to generate spectral libraries for targeted method development in Skyline. PECAN correlates a list of peptide sequences with the acquired DIA spectra in order to locate peptide-specific spectra within the acquired DIA dataset. The PECAN .blib file was imported into Skyline to select peptide transitions and create MS methods that would target specific peptides and transitions in a SRM assay.
Corresponding dataset(s) in other omics resources
Timmins-Schiffman EB, Crandall GA, Vadopalas B, Riffle ME, Nunn BL, Roberts S. Integrating discovery-driven proteomics and selected reaction monitoring to develop a non-invasive assay for geoduck reproductive maturation. J Proteome Res. 2017 Jul 21 PubMed: 28730805