Evaluation of RNAlater™ as a field-compatible preservation method for metaproteomic analyses of bacteria-animal symbioses

Abstract

Field studies are central to environmental microbiology and microbial ecology as they enable studies of natural microbial communities. Metaproteomics, the study of protein abundances in microbial communities, allows to study these communities in situ which requires protein preservation directly in the field as protein abundance patterns can change rapidly after sampling. Ideally, a protein preservative for field deployment works rapidly and preserves the whole proteome, is stable in long-term storage, is non-hazardous and easy to transport, and is available at low cost. Although these requirements might be met by several protein preservatives, an assessment of their suitability in field conditions when targeted for metaproteomics is currently lacking. Here, we compared the protein preservation performance of flash freezing and the preservation solution RNAlater using the marine gutless oligochaete Olavius algarvensis and its symbiotic microbes as a test case. In addition, we evaluated long-term RNAlater storage after 1 day, 1 week and 4 weeks at room temperature (22-23 {degrees}C). We evaluated protein preservation using one dimensional liquid chromatography tandem mass spectrometry (1D-LC-MS/MS). We found that RNAlater and flash freezing preserved proteins equally well in terms of total number of identified proteins or relative abundances of individual proteins and none of the test time points were altered compared to t0. Moreover, we did not find biases against specific taxonomic groups or proteins with particular biochemical properties. Based on our metaproteomics data and the logistical requirements for field deployment we recommend RNAlater for protein preservation of field-collected samples when targeted for metaproteomcis. ImportanceMetaproteomics, the large-scale identification and quantification of proteins from microbial communities, provides direct insights into the phenotypes of microorganisms on the molecular level. To ensure the integrity of the metaproteomic data, samples need to be preserved immediately after sampling to avoid changes in protein abundance patterns. In laboratory set-ups samples for proteomic analyses are most commonly preserved by flash freezing; however, liquid nitrogen or dry ice is often unavailable at remote field locations due to its hazardous nature and transport restrictions. Our study shows that RNAlater can serve as a low hazard, easy to transport alternative to flash freezing for field preservation of samples for metaproteomics. We show that RNAlater preserves the metaproteome equally well as compared to flash freezing and protein abundance patterns remain stable during long-term storage for at least 4 weeks at room temperature.