Quantifying Protein Acetylation in Diabetic Nephropathy from FFPE Explants

ABSTRACT: Background: Diabetic kidney disease (DKD) is a serious complication of diabetes mellitus and a leading cause of chronic kidney disease and end stage renal disease. One potential mechanism underlying cellular dysfunction contributing to kidney disease is aberrant post-translational modifications, such as lysine acetylation. Lysine acetylation is associated with cellular metabolic flux and is thought to be altered in patients with diabetes and dysfunctional renal metabolism. Methods: A novel technique was adapted to quantify sites of N-acetylation from formalin-fixed paraffin-embedded kidney explant tissue from patients with diabetic kidney disease and non-diseased donors (n=5 and n=7, respectively). Multiple methodologies were integrated to extract viable proteins from formalin-fixed paraffin-embedded kidney explant tissue followed by enrichment and isolation. Proteomic and acetylomic profiles were then quantified via LC-MS/MS analysis. Results: Quantitative proteomic analysis of FFPE tissues identified 840 total proteins with 260 of those significantly changing and our acetylomic analysis quantified 290 acetylated peptides with 98 of those significantly changing in human diabetic kidneys. Lysine acetylation is known to regulate protein function, providing a mechanism by which proteins respond to cellular metabolic status. Protein pathways found to be impacted in DKD patients revealed an association with numerous metabolic pathways, specifically mitochondrial function, EIF2 signaling, oxidative phosphorylation, lipid metabolism, sirtuin signaling, and LXR/RXR activation, each of which are intimated to play a significant role in the pathogenesis of DKD. Differential protein acetylation in DKD patients reflected biochemical processes associated with sirtuin signaling, valine, leucine, and isoleucine degradation, lactate metabolism, oxidative phosphorylation, and ketogenesis. Conclusions: Here, we establish a quantitative acetylomics platform for protein biomarker discovery in formalin-fixed and paraffin-embedded biopsies of kidney transplant patients suffering from diabetic kidney disease. Our analysis provides a novel platform for quantifying enriched post-translational modifications in preserved explant tissues and includes optimizations for addressing protein crosslinking, paraffin removal, and formalin-derived formaldehyde protein adducts combined with immunoprecipitation of acetyl-peptides and total protein quantitation.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Kidney

DISEASE(S): Diabetes Mellitus

SUBMITTER: Cole Michel  

LAB HEAD: Kristofer Fritz

PROVIDER: PXD041884 | Pride | 2025-11-10

REPOSITORIES: Pride