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Abstract
Improvements in temporal and spatial sampling frequency have the potential to open new windows into the understanding of marine microbial dynamics. In recent years, efforts have been made to allow automated samplers to collect microbial biomass for DNA/RNA analyses from moored observatories and autonomous underwater vehicles. Measurements of microbial proteins are also of significant interest given their biogeochemical importance as enzymes that catalyze reactions and transporters that interface with the environment. We examined the influence of five preservatives solutions (SDS-extraction buffer, ethanol, trichloroacetic acid, B-PER, and RNAlater) on the proteome integrity of the marine cyanobacterium Synechococcus WH8102 after 4 weeks of storage at room temperature. Four approaches were used to assess degradation: total protein recovery, band integrity on an SDS detergent polyacrylamide electrophoresis (SDS-PAGE) gel, and number of protein identifications and relative abundances by 1-dimensional LC–MS/MS proteomic analyses. Total protein recoveries from the preserved samples were lower than the frozen control due to processing losses, which could be corrected for with internal standardization. The trichloroacetic acid preserved sample showed significant loss of protein band integrity on the SDS-PAGE gel. The RNAlater preserved sample showed the highest number of protein identifications (103% relative to the control; 520 ± 31 identifications in RNAlater versus 504 ± 4 in the control), equivalent to the frozen control. Relative abundances of individual proteins in the RNAlater treatment were quite similar to that of the frozen control (average ratio of 1.01 ± 0.27 for the 50 most abundant proteins), while the SDS-extraction buffer, ethanol, and B-PER all showed significant decreases in both number of identifications and relative abundances of individual proteins. Based on these findings, RNAlater was an effective proteome preservative, although further study is warranted on additional marine microbes.
Highlights
It is anticipated that higher spatial and temporal sampling of the oceans provided by deployment of a combination of in situ sensors and autonomous sample collectors will greatly improve our understanding of marine processes
For the purposes of this degradation study, highly robust, and repeatable protein identification numbers and spectral counts (SpC) were found with 1-D chromatography (Saito et al, 2011), and this approach was more useful for our immediate the needs of this study than the deeper proteome depth allowed by multi-dimensional chromatography, which we have found can be more variable in both number of protein identifications and spectral counts for technical replicates
Four techniques were used to examine protein recovery after extraction: total protein concentration, a qualitative examination of band integrity on 1-D-SDS-PAGE gel, total number of proteins identified by LC–MS, and ratios of relative abundance of each individual protein between each preservative and the control as determined by spectral counting
Summary
It is anticipated that higher spatial and temporal sampling of the oceans provided by deployment of a combination of in situ sensors and autonomous sample collectors will greatly improve our understanding of marine processes This is likely to be true for coupled microbiological and chemical processes that scale from genomic potential to global biogeochemical impacts on virtually all biologically utilized elements (Morel and Price, 2003; Falkowski et al, 2008; Saito et al, 2008). Using isotopically labeled peptide standards, it is possible to measure very low quantities of proteins on an absolute scale This has significant potential for application to oceanographic biogeochemical studies where concentrations of key biogeochemical enzymes can be quantified and using their measured ranges of activities from laboratory studies, estimates of potential in situ biogeochemical reaction rates could be calculated for environmental samples (Bertrand et al, 2011; Saito et al, 2011). A culture of marine cyanobacterium Synechococcus WH8102 was used for model proteinaceous material because of the ubiquity and abundance of this and other marine cyanobacteria in the oceans (Waterbury et al, 1986; Partensky et al, 1999; Saito et al, 2005)
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