ZenoSWATH proteomics and clustering analysis reveal different modulation patterns in fibroblasts from cystinosis patients by cysteamine

Introduction

Cystinosis is a rare metabolic disease, where mutations in the CTNS gene, which codifies for a symporter that mediates export of cystine from lysosomes, result in the accumulation of cystine crystals in the cells. The metabolic imbalance caused by intra-lysosomal cystine accumulation potentially leads to serious complications in various organs, especially kidneys, eyes and muscles, eventually leading to kidney transplantation. Up to one-third of the patients die before age 30 if untreated.

Cysteamine (CTA) therapy, which reduces cystine levels, is the primary treatment for cystinosis. CTA ameliorates the symptoms and delays the progression, improving patient quality of life. But it does not cure the disease, has serious undesirable side effects, and fails in preventing the end-stage renal failure. Thus, it is crucial to characterize the cellular events occurring in the affected cells, identifying the mechanisms involved in both the beneficial and adverse effects of cysteamine.

Methodology

Skin fibroblasts from cystinosis patients and healthy controls were cultured with increasing concentrations of CTA (0, 15, 30, 75, 150 and 300 µM). Cells were lysated and the protein samples were prepared using the iST kit (PreOmics). 400 ng of the resulting peptidic digests were loaded on Evotips (Evosep) and analyzed by LC-MS in a ZenoTOF 7600 (SCIEX) coupled to a Evosep One (Evosep). A 60 SPD method (21 min total run time), using an 8 cm x 150 μm Performance column (Evosep) was used for LC separation. A variable ZenoSWATH DIA method, was used, combining a 25 ms TOF MS scan with 63 20 ms MS/MS scans in variable isolation windows (range 400-991 m/z). The ZenoSWATH runs were processed in a library-free workflow using DIA-NN v1.8.1, and the quantitative profiles of the quantified proteins were analyzed using VSClust v1.2 for obtaining the protein clusters that showed similar expression profiles. For functional analysis, STRING v12.0 and Metascape v3.5 were used for obtaining the detailed pathway and process enrichment for each protein cluster.

Results

Six protein clusters exhibiting similar expression profiles across the increasing CTA concentrations were identified by VSClust after ZenoSWATH quantitation. Interestingly, for a great number of the protein changes found in cystinotic versus non-cystinotic patients, CTA is not reversing these changes, and in some case is even exacerbating these differences, providing an explanation for the shortcomings and the side effects in the therapeutic action of CTA. However, for some proteins CTA does seem to correct the effect of the disease, and we report the potential cellular mechanisms by which this beneficial effect of CTA could take place.