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Comparison of extracellular matrix enrichment protocols for the improved characterization of the skin matrisome by mass spectrometry

ABSTRACT: Four protocols were adapted and compared to enrich extracellular matrix proteins from mouse skin: 1. compartmental enrichment; 2. chemical decellularization; 3. enzymatic decellularization using phospholipase A2; 4. quantitative detergent solubility profiling (QDSP). All four protocols were started the same day, each using 35 mg of frozen back skin taken from the same initial back skin piece. The experiment was repeated three times starting from three different mice. Compartmental enrichment: skin samples were processed using the CNMCS commercial kit. Briefly, the skin tissue was homogenized in buffer C and successively incubated under agitation in buffers W, N, M and CS supplemented with the protease inhibitors from the kit. Extraction buffer C and insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. Chemical decellularization: skin samples were cut into small pieces and successively incubated in buffers containing NaCl, SDS/sodium deoxycholate, Triton X-100 and GuHCl. Extractions buffers as well as the final insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. Enzymatic decellularization: skin samples were cut into small pieces and successively incubated in buffers containing NaCl, phosopholipase A2/sodium deoxycholate. Extraction buffers as well as the insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. QDSP: skin samples were homogenized in sterile PBS and successively incubated in 3 buffers containing increasing concentrations in detergents (including SDS, sodium deoxycholate, NP-40) as previously described. Extraction buffers as well as the insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. For comparison with the four protocols described above, total skin lysates were also prepared for two of the mice. In this case, 20 mg of mouse skin were homogenized and incubated in a lysis buffer containing SDS, β-mercaptoethanol. Samples were centrifuged and the supernatant was kept aside, flash-frozen in liquid nitrogen immediately.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Skin

SUBMITTER: Frederic DELOLME

LAB HEAD: Catherine Moali

PROVIDER: PXD025842 | Pride | 2021-11-02

REPOSITORIES: Pride

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			Action	DRS
	200717-MD-0372-0373-Trembl.mzML	Mzml
	200717-MD-0374-0376-Trembl.mzML	Mzml
	200717-MD-0378-0381-Trembl_new.mzML	Mzml
	200825-MD-0400-0401-Trembl.mzML	Mzml
	201211-MD-Mouse-1_Det.mzML	Mzml

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Project description:BACKGROUND: Stool metabolites provide essential insights into the function of the gut microbiome. The current gold standard for storage of stool samples for metabolomics is flash-freezing at - 80 °C which can be inconvenient and expensive. Ambient temperature storage of stool is more practical, however no available methodologies adequately preserve the metabolomic profile of stool. A novel sampling kit (OMNImet.GUT; DNA Genotek, Inc.) was introduced for ambient temperature storage and stabilization of feces for metabolomics; we aimed to test the performance of this kit vs flash-freezing. To do this stool was collected from an infant's diaper was divided into 2 aliquots: 1) flash-frozen and 2) stored in an OMNImet.GUT tube at ambient temperature for 3-4 days. Samples from the same infant were collected at 2 different timepoints to assess metabolite changes over time. Subsequently, all samples underwent metabolomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).RESULTS: Paired fecal samples (flash-frozen and ambient temperature) from 16 infants were collected at 2 timepoints (32 individual samples, 64 aliquots). Similar numbers of metabolites were detected in both the frozen and ambient temperature samples (1126 in frozen, 1107 in ambient temperature, 1064 shared between sample types). Metabolite abundances were strongly correlated between storage methods (median Spearman correlation Rs = 0.785 across metabolites). Hierarchical clustering analysis and principal component analysis showed that samples from the same individuals at a given timepoint clustered closely, regardless of the storage method. Repeat samples from the same individual were compared by paired t-test, separately for the frozen and OMNImet.GUT. The number of metabolites in each biochemical class that significantly changed (p < 0.05) at timepoint 2 relative to timepoint 1 was similar in flash-frozen vs ambient temperature storage. Changes in microbiota modified metabolites over time were also consistent across both methodologies.CONCLUSION: Ambient temperature storage and stabilization of stool in the OMNImet.GUT device yielded comparable metabolomic results to flash freezing in terms of 1) the identity and abundance of detected biochemicals 2) the distinct metabolomic profiles of subjects and 3) changes in metabolites over time that are plausibly microbiota-induced. This method potentially provides a more convenient, less expensive home collection and storage option for stool metabolomic analysis.

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Comparison of extracellular matrix enrichment protocols for the improved characterization of the skin matrisome by mass spectrometry

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