Project description:When studying gene expression in microbe-animals symbioses collected in the field it is essential to quickly and efficiently preserve in situ symbiont and host gene expression patterns. One of the most commonly used sample preservation methods for samples targeted for proteomic analyses is flash freezing, however, liquid nitrogen or dry ice needed for flash freezing are often not available at remote field sites. We tested if RNAlater allows to preserve proteins in animal-microbe symbioses as efficiently as flash freezing and without introducing issues with downstream processing. We used the marine gutless oligochaete Olavius algarvensis as a model for testing. Olavius algarvensis lives in shallow water sediments off the coast of Elba, Italy. It has no digestive and excretory system and harbors five bacterial symbionts that fulfill its nutritional and waste recycling needs (Kleiner et al., 2012, PNAS 109(19):1173-82). We compared five RNAlater preserved and five flash frozen samples in terms of the number of identified proteins, abundances of individual proteins and potential biases against specific protein or taxonomic groups. Five worms were incubated in RNAlater for 24 hours. After incubation, RNAlater was removed and samples were stored at -80°C. The remaining five worms were preserved with liquid nitrogen and stored at -80 °C immediately after preservation.
Project description:When studying gene expression in microbe-animals symbioses collected in the field it is essential to quickly and efficiently preserve in situ symbiont and host protein abundance patterns. One of the most commonly used sample preservation methods for samples targeted for proteomic analyses is flash freezing, however, liquid nitrogen or dry ice needed for flash freezing are often not available at remote field sites. We replicated our experiment from PXD014591 to test if RNAlater allows preserving proteins in animal-microbe symbioses as efficiently as flash freezing and without introducing issues with downstream processing. We used the marine gutless oligochaete Olavius algarvensis as a model for testing. Olavius algarvensis lives in shallow water sediments off the coast of Elba, Italy. It has no digestive and excretory system and harbors five bacterial symbionts that fulfill its nutritional and waste recycling needs (Kleiner et al., 2012, PNAS 109(19):1173-82). We compared six RNAlater preserved and eight flash frozen samples in terms of the number of identified proteins, abundances of individual proteins and potential biases against specific protein or taxonomic groups. Six worms were incubated in RNAlater for 24 hours. After incubation, RNAlater was removed and samples were stored at -80°C. Eight worms were directly flash frozen in liquid nitrogen and stored at -80 °C immediately after preservation.
Project description:We developed a method that allows measuring the stable carbon isotope composition of individual species in microbial communities using metaproteomics. We call this methods “Direct Protein-SIF”. We validated and tested the method extensively using pure cultures (PXD006762) and mock communities (PXD006118, https://www.ebi.ac.uk/pride/archive/projects/PXD006118). As a case study we applied Direct Protein-SIF to the Olavius algarvensis symbiosis. Olavius algarvensis is a marine worm that lives in shallow-water sediments off the coast of Elba, Italy. The worm has no digestive and excretory system, instead it harbors five bacterial symbionts that fulfill its nutritional and waste recycling needs (http://www.pnas.org/content/109/19/E1173.short). For this project we generated metaproteomes of 14 O. algarvensis individuals. A total of 18 LC-MS/MS runs were generated. For Direct Protein-SIF the data from all runs was combined. In this submission we are including the Direct Protein-SIF specific isotope pattern file as well as the .mzML files and PSM file required as input for the Direct Protein-SIF software (Calis-p). In addition to the Olavius algarvensis samples a protein reference material (MKH files) was measured. The respective .raw files and isotopic pattern files are available through project PXD006762 (see publication for details on how the reference material is used to calibrate the method).
Project description:Transcriptomic and proteomic insights into innate immunity and adaptations to a symbiotic lifestyle in the gutless marine worm Olavius algarvensis
Project description:When studying gene expression in microbe-animals symbioses collected in the field it is essential to quickly and efficiently preserve in situ symbiont and host gene expression patterns. One of the most commonly used sample preservation methods for samples targeted for proteomic analyses is flash freezing, however, liquid nitrogen or dry ice needed for flash freezing are often not available at remote field sites. We first tested if RNAlater allows to preserve proteins in animal-microbe symbioses as efficiently as flash freezing and without introducing issues with downstream processing (see PXD014591). Second, for the data in this PRIDE submission we tested if RNAlater preserves protein expression patterns over time at room temperature. We used the marine gutless oligochaete Olavius algarvensis as a test case. Olavius algarvensis lives in shallow water sediments off the coast of Elba, Italy. It has no digestive and excretory system and harbors five bacterial symbionts that fulfill its nutritional and waste recycling needs (Kleiner et al., 2012, PNAS 109(19):1173-82). For this dataset, we fixed a total of 33 worms and incubated them in RNAlater for up to 4 weeks. We then evaluated proteome preservation quality in terms of the number of identified proteins, abundances of individual proteins and potential biases against specific protein or taxonomic groups. Out of this 33 samples, eleven worms were incubated for 24 hours in RNAlater at 4°C (t0), while the other worms were incubated in RNAlater at room temperature (21-23°C) for additional 24 hours (t1, 6 worms), one week (t2, 8 worms), and four weeks (t3, 8 worms). We removed RNAlater from the worms after incubation and froze the samples at -80°C.