Assessing the impact of long term frozen storage of faecal samples on protein concentration and protease activity

Abstract

BackgroundThe proteome is the second axis of the microbiome:host interactome and proteases are a significant aspect in this interaction. They interact with a large variety of host proteins and structures and in many situations are implicated in pathogenesis. Furthermore faecal samples are commonly collected and stored frozen so they can be analysed at a later date. So we were interested to know whether long term storage affected the integrity of proteases and total protein and whether historical native faecal samples were still a viable option for answering research questions around the functional proteome.MethodsFaecal samples were collected from 3 healthy volunteers (3 biological replicates) and processed in order to be stored at both − 20 °C and − 80 °C and in a variety of storage buffers. Protein extraction, protein content and protease activity were assessed at the time of collection, after 24 h, 1 week, 1 month, 3 months 6 months and finally 1 year.ResultsBeadbeating impacted the quantity of protein extracted, while sodium azide did not impact protease assays. Long term storage of extracted proteins showed that both total protein and protease activity were affected when they were stored as extracted protein. Intact faecal samples were shown to maintain both protein levels and protease activity regardless of time and temperature.ConclusionsBeadbeating increases the protein and protease activity when extracting from a faecal sample, however, the extracted protein is not stable and activity is lost, even with a suitable storage buffer. The most robust solution is to store the proteins in an intact frozen native faecal matrix and extract at the time of assay or analysis, this approach was shown to be suitable for samples in which, there are low levels of protease activity and which had been frozen for a year.

Effects of the long-term storage of human fecal microbiota samples collected in RNAlater

“…We aimed here simply to evaluate the effect of the long-term storage of stool samples destined for processing for 16S rRNA gene sequencing, but our findings suggest that long-term storage may influence samples stored for metagenomics, particularly for studies at higher levels of resolution (e.g. at strain level), transcriptomic, metabolomic or proteomic studies 33,43 . Depending on the final use, methods of sample collection and preservation other than those described here may be used, with potentially different impacts on microbiota composition.…”

Ursodeoxycholic acid enriches intestinal bile salt hydrolase-expressing Bacteroidetes in cholestatic pregnancy

“…Bile salt hydrolase activity assay. Faecal water was prepared and total faecal protein quantified similarly to a method previously-described 41 , but with the addition of bacterial and mammalian protease inhibitor cocktails (G Biosciences, Uttar Pradesh, India), as well as Dithiothreitol to 1 mM final concentration (Roche, Basel, Switzerland) (to minimise enzyme oxidation 42 ).…”

Age-related levels of fecal M2-pyruvate kinase in children with cystic fibrosis and healthy children 0 to 10 years old

“…In addition, the stability of long term frozen storage of fecal M2-PK needs evaluation. A previous study evaluating protein degradation in fecal samples stored intact (rather than as extracted proteins) showed that proteins remained stable in samples frozen at both − 20 and − 80 degrees Celsius after 24 h, 1 week, 1 month, 3 months, 6 months and 1 year [34]. There was a significant difference in age between CF and HC subjects at sample collection, however it was demonstrated that fecal M2-PK levels were independent of age using linear mixed model analysis.…”