| Metadata |
| datasetIdentifier | PASS00917 |
| datasetType | SRM |
| submitter | Emma Timmins-Schiffman <emmats@uw.edu> |
| submitter_organization | University of Washington |
| lab_head_full_name | Brook Nunn |
| lab_head_email | brookh@uw.edu |
| lab_head_organization | University of Washington |
| lab_head_country | United States |
| datasetTag | OceanBacteriaSRM |
| datasetTitle | Detection of Low Abundance Ocean Bacterial Peptides SRM |
| publicReleaseDate | 2016-08-08 00:00:00 |
| finalizedDate | 2016-08-08 13:17:52 |
| summary | Bacteria transform nutrients and degrade organic matter, making them an essential part of healthy ecosystems. By assaying bacterial physiology within a complex system, the status of the whole ecosystem can be investigated. Proteins are the dynamic molecules that control essential bacterial physiological responses and those of every organism; characterizing an organism’s proteome can therefore provide information on its interaction with the environment. Data dependent proteomic analysis (DDA) is a global approach to assay the entire proteome, but sample complexity and the stochastic nature of mass spectrometry can make it difficult to detect low abundance proteins. We explored the development of targeted proteomic (selected reaction monitoring, SRM) assays in complex ocean samples in order to detect specific bacterial proteins of interest and to assess new tools for mixed community metaproteomic exploration. A mixed community was created from a dilution series of an isolated cultures of bacteria (Reugeria pomeroyi) and phytoplankton (Thalassiosira pseudonana). Using SRM, we were able to select and detect bacterial peptides from the community that were undetectable with the standard DDA approach. We demonstrate benefits and drawbacks of different proteomic approaches that can be used to probe for and resolve nuances of bacterial physiological processes in complex environmental systems. |
| contributors | Emma Timmins-Schiffman, Brook Nunn, Rodger Harvey, Molly Mikan, Ying Sonia Ting |
| publication | unpublished |
| growth | Ruegeria pomeroyi (Rpom, NCMA B3) was reconstituted in autoclaved and filtered 0.5 YTSS media and slowly transitioned into a lower glucose medium of 0.625 mM over multiple generations. Cultures were grown under axenic conditions at room temperature and bacterial growth was tracked with absorbance measurements at 600 nm. The culture was harvested during stationary phase. |
| treatment | |
| extraction | Proteins were extracted from filters following Nunn et al. (2015). |
| separation | |
| digestion | |
| acquisition | 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 SRM experiments included 1 µg protein plus the internal standard in a 3 µl injection. A C18 trap (2 cm) and C18 analytical columns (27.5 cm) were used and each sample was analyzed in 2 MS experiments to cover the entire peptide transition list (n=334). Transitions were acquired over a 40 minute gradient of 2-60% acetonitrile. |
| informatics | Acquired SRM data were analyzed in Skyline. A Skyline document was prepared with a list of peptide transitions used to generate the method file for SRM data acquisition and can be found on Panorama, panoramaweb.org. Peptide transition MS2 peaks were quantified using peak area integration across all samples. Peak presence was determined based on consistency of retention time (verified by spiked in QC peptides) and peak morphology. |
| instruments | Thermo Vantage |
| species | Ruegeria pomeroyi |
| massModifications | none |
