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Sample Preservation and Storage Significantly Impact Taxonomic and Functional Profiles in Metaproteomics Studies of the Human Gut Microbiome.

Author(s): Hickl O, Heintz-Buschart A, Trautwein-Schult A, Hercog R, Bork P, Wilmes P, Becher D

Publication: Microorganisms, 2019, Vol. 7, Page

PubMed ID: 31546776 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the effects of preservation by snap-freezing in liquid nitrogen and RNAlater on the detection of microbial proteins and nucleic acids in fecal specimens. Stool specimens were collected in MedAuxil stool collectors, aliquoted and snap-frozen in liquid nitrogen (150 mg) or stored in RNAlater at 4°C for 6 h (200 mg). Aliquots were then stored at -80°C for an unspecified duration. Ambion RNAlaterICE was added to snap-frozen aliquots and specimens were incubated for 16 h, homogenized, and then biomolecules extracted using the AllPrep DNA/RNA/Protein Kit. RNAlater aliquots were thawed on ice, homogenized, and extraction performed using the AllPrep DNA/RNA/Protein Kit. Proteins were separated by gel electrophoresis, digested with trypsin, eluted, desalted with ZipTip-tips, and analyzed by High-Pressure Liquid Chromatography and Mass Spectrometry. DNA and RNA libraries were prepared using a dual barcoding system and sequenced on an Illumina HiSeq 4000 and Illumina NextSeq 500.

   Summary of Findings:

   The majority of the 2000 bacterial proteins identified as significantly differentially abundant between snap-frozen and RNAlater-preserved specimens were of clostridial origin (~65% and 55%, respectively), 1300 of these were in higher abundance in snap-frozen specimens than RNAlater-preserved specimens (P<0.05). Actinobacteria proteins comprised 35% of proteins found at higher abundance in snap-frozen specimens compared to only ~0.2% of the proteins found at higher abundance in RNAlater-preserved specimens. In contrast, 30% of the significantly more abundant proteins in RNAlater-preserved specimens were Bacteroidia but only 1.5% in snap-frozen specimens. Similarly, almost no Proteobacteria and Negativicutes were identified in snap-frozen specimens but made up ~5% and 10% of the proteins in RNAlater specimens, respectively. Metagenomics analysis largely concurred with the proteomics analysis; however, Bacteroidia was more abundant in the metagenomics analysis and Clostridia made up a larger share in metaproteomics.

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • RNAlater
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing
   Protein	MS/MS
   Protein	HPLC
   RNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Snap frozen RNAlater

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