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Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization

ABSTRACT: Four single consecutive murine kidney tissue specimens were prepared with the direct trypsinization with RapiGest (DTR) approach or with the filter aided sample preparation (FASP) protocol using both 10k and 30k filter devices and analyzed by label-free, quantitative liquid chromatography – tandem mass spectrometry (LC-MS/MS). Furthermore, we probed compatibility of the DTR protocol for the analysis of common histological stainings, namely hematoxylin & eosin (H&E), hematoxylin, and hemalaun. These were proteomically compared to an unstained control by analyzing four human tonsil FFPE tissues per condition.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Tonsil, Kidney

SUBMITTER: Melanie Christine Föll

LAB HEAD: Oliver Schilling

PROVIDER: PXD006401 | Pride | 2018-08-15

REPOSITORIES: Pride

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Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization.

Föll Melanie Christine MC Fahrner Matthias M Oria Victor Oginga VO Kühs Markus M Biniossek Martin Lothar ML Werner Martin M Bronsert Peter P Schilling Oliver O

Clinical proteomics 20180306

<h4>Background</h4>Proteomic analyses of clinical specimens often rely on human tissues preserved through formalin-fixation and paraffin embedding (FFPE). Minimal sample consumption is the key to preserve the integrity of pathological archives but also to deal with minimal invasive core biopsies. This has been achieved by using the acid-labile surfactant RapiGest in combination with a direct trypsinization (DTR) strategy. A critical comparison of the DTR protocol with the most commonly used filt ...[more]

PMID: 29527141

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Project description:We aimed to identify miRNA biomarkers of renal injury in kidney biopsies from patients with lupus nephritis. MiRNA profiles of 8 patients were analyzed for correlation with various clinical features including Progression, Activity, Chronicity, and Time to Kidney Failure. MicroRNAs (miRs) are promising biomarkers and are involved in pathogenesis of kidney diseases. We aimed to identify miR biomarkers of renal injury in kidney biopsies from patients with lupus nephritis and study their potential role in renal fibrosis. miR-150 was significantly increased in kidneys with high chronicity compared to low chronicity and it correlated positively with chronicity index scores and renal collagen I expression. In kidneys with high chronicity, miR-150 was found predominantly in proximal tubular cells (PTCs) and was moderately expressed in podocytes and to lesser degree in mesangial cells (MCs). We hypothesized that miR-150 increases fibrosis by downregulating a negative regulator of profibrotic proteins. Suppressor of cytokine signaling1 (SOCS1) is a predicted target of miR-150 and has shown antifibrotic role. After confirming that SOCS1 is a direct target of miR-150, we showed that transfection of a miR-150 analog downregulated SOCS1 protein and upregulated the profibrotic proteins fibronectin, collagen I, collagen III, and TGF-β1 in both primary normal human renal PTCs and MCs. A similar effect was seen when using a SOCS1 siRNA to confirm that the effect of miR-150 on profibrotic proteins is mediated through SOCS1. Stimulation with TGF-β1 induced miR-150 increase in PTCs and human podocytes but not MCs. These results suggest that miR-150 might be a useful quantitative renal biomarker of kidney injury in lupus nephritis and that miR-150, which might be partially induced by TGF-β1, plays an important role in renal fibrosis by increasing profibrotic molecules through downregulation of SOCS1. FFPE kidney specimens (n=25) including baseline and repeated needle renal biopsies were from 14 patients with LN enrolled in IRB-approved protocols at the NIDDK between 1976 and 1999. The specimens were divided in two groups based on histological chronicity index (CI). CI ≥ 4 were categorized as having high degree of chronicity of chronic kidney injury. 18 kidneys from 8 patients including high CI (n=9) and low CI (n=9) were used for miR profiling by Affymetrix microRNA microarrays.

			Action	DRS
	10_FASP30k_2.raw	Raw
	10_HE2_2.raw	Raw
	11_FASP30k_3.raw	Raw
	11_hematoxylin2_1.raw	Raw
	12_FASP30k_4.raw	Raw