title: Assessing the impact of long term frozen storage of faecal samples on protein concentration and protease activity.
abstract: BACKGROUND
  The 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.
  
  
  METHODS
  Faecal 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 24h, 1 week, 1 month, 3 months 6 months and finally 1 year.
  
  
  RESULTS
  Beadbeating 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.
  
  
  CONCLUSIONS
  Beadbeating 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.
authors: School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK; College of Medicine, Swansea University, Swansea SA2 8PP, UK.
authors: Laura S
authors: LS
authors: Morris
authors: School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK; Centre for Digestive and Gut Health, Imperial College London, London, UK. Electronic address: marchesijr@cardiff.ac.uk.
authors: Julian R
authors: JR
authors: Marchesi
mesh_terms: D005243:Feces / Q000737:chemistry* / Q000382:microbiology; D006801:Humans; D008297:Male; D064307:Microbiota; D010447:Peptide Hydrolases / Q000032:analysis; D011309:Preservation, Biological / Q000295:instrumentation / Q000379:methods*; D011506:Proteins; D013696:Temperature; D013997:Time Factors; D055815:Young Adult
chemical_list: D011506:Proteins; D010447:Peptide Hydrolases
keywords: Enzyme activity; Gut; Long-term storage; Microbiota; Proteases; Proteins