Standard-flow LC and thermal focusing ESI elucidates altered liver proteins in late stage Niemann-Pick, type C1 disease.

Aim: Mass spectrometry (MS)-based proteomics, particularly with the development of nano-ESI, have been invaluable to our understanding of altered proteins related to human disease. Niemann-Pick, type C1 (NPC1) disease is a fatal, autosomal recessive, neurodegenerative disorder. The resulting defects include unesterified cholesterol and sphingolipids accumulation in the late endosomal/lysosomal system resulting in organ dysfunction including liver disease. Materials & methods: First, we performed MS analysis of a complex mammalian proteome using both nano- and standard-flow ESI with the intent of developing a differential proteomics platform using standard-flow ESI. Next, we measured the differential liver proteome in the NPC1 mouse model via label-free quantitative MS using standard-flow ESI. Results: Using the standard-flow ESI approach, we found altered protein levels including, increased Limp2 and Rab7a in liver tissue of Npc1-/- compared to control mice. Conclusion: Standard-flow ESI can be a tool for quantitative proteomic studies when sample amount is not limited. Using this method, we have identified new protein markers of NPC1.

Quantitative, Label-Free Proteomics in the Symptomatic Niemann-Pick, Type C1 Mouse Model Using Standard Flow Liquid Chromatography and Thermal Focusing Electrospray Ionization.

Niemann-Pick disease, type C1 (NPC1) is a fatal, autosomal recessive, neurodegenerative disorder caused by mutations in the NPC1 gene. As a result, there is accumulation of unesterified cholesterol and sphingolipids in the late endosomal/lysosomal system. This abnormal accumulation results in a cascade of pathophysiological events including progressive, cerebellar neurodegeneration, among others. While significant progress has been made to better understand NPC1, the downstream effects of cholesterol storage and the major mechanisms that drive neurodegeneration remain unclear. In the current study, a) the use of a commercial, highly efficient standard flow-ESI platform for protein biomarker identification is implemented and b) protein biomarkers are identified and evaluated at a terminal time point in the NPC1 null mouse model. In this study, alterations are observed in proteins related to fatty acid homeostasis, calcium binding and regulation, lysosomal regulation, and inositol biosynthesis and metabolism, as well as signaling by Rho family GTPases. New observations from this study include altered expression of Pcp2 and Limp2 in Npc1 mutant mice relative to control, with Pcp2 exhibiting multiple isoforms and specific to the cerebella. This study provides valuable insight into pathways altered in the late-stage pathophysiology of NPC1.