Quantification of cystine in human renal proximal tubule cells using liquid chromatography–tandem mass spectrometry

Abstract

Nephropathic cystinosis is characterized by abnormal intralysosomal accumulation of cystine throughout the body, causing irreversible damage to various organs, particularly the kidneys. Cysteamine, the currently available treatment, can reduce lysosomal cystine and postpone disease progression. However, cysteamine poses serious side effects and does not address all of the symptoms of cystinosis. To screen for new treatment options, a rapid and reliable high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) method was developed to quantify cystine in conditionally immortalized human proximal tubular epithelial cells (ciPTEC). The ciPTEC were treated with N‐ethylmaleimide, lysed and deproteinized with 15% (w/v) sulfosalicylic acid. Subsequently, cystine was measured using deuterium‐labeled cystine‐D4, as the internal standard. The assay developed demonstrated linearity to at least 20 μmol/L with a good precision. Accuracies were between 97.3 and 102.9% for both cell extracts and whole cell samples. Cystine was sufficiently stable under all relevant analytical conditions. The assay was successfully applied to determine cystine levels in both healthy and cystinotic ciPTEC. Control cells showed clearly distinguishable cystine levels compared with cystinotic cells treated with or without cysteamine. The method developed provides a fast and reliable quantification of cystine, and is applicable to screen for potential drugs that could reverse cystinotic symptoms in human kidney cells.

Gas-Phase Fragmentation Reactions of Protonated Cystine using High-Resolution Tandem Mass Spectrometry

“…Its levels in biological samples are commonly measured by SRM. The SRM transition 241→74 [ 4 , 10 , 12 , 13 , 22 ] or 241→152 [ 6 , 7 , 8 , 9 , 11 , 22 ] is mostly used for cystine quantification to increase sensitivity. Our data showed that the m/z 152 fragment ion was predominant when the collision energy was low ( Figure 2 a).…”

“…Cystine in urine, blood, or other biological samples has been used as an important biomarker for various pathological conditions, such as inherited metabolic disorders and cystinosis [ 4 , 5 , 6 ]. Quantification of cystine in leukocytes and urine by CID-based LC–MS/MS is performed in the clinical routine for diagnosis of nephropathic cystinosis [ 7 , 8 , 9 , 10 ]. Selected reaction monitoring (SRM) is the mostly reported method for measuring cystine [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ].…”

“…Quantification of cystine in leukocytes and urine by CID-based LC–MS/MS is performed in the clinical routine for diagnosis of nephropathic cystinosis [ 7 , 8 , 9 , 10 ]. Selected reaction monitoring (SRM) is the mostly reported method for measuring cystine [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. Thus, a good knowledge of the gas-phase fragmentation chemistry of cystine in CID is essential to design MS/MS-based assays for reliable cystine testing.…”

Cysteamine–bicalutamide combination therapy corrects proximal tubule phenotype in cystinosis

“…In agreement with the increased TFEB activity, bicalutamide also restored endocytic cargo processing in cystinotic cells (Fig 5D and E), allowing us to test whether the increased degradation of lysosomal cargo results in decreased cystine accumulation. We used a more sensitive and quantitative LC-MS/MS method (Jamalpoor et al, 2018) to measure cystine levels in cystinotic cells upon treatment with cysteamine, bicalutamide, or their combination. In line with metabolomics data, CTNS À/À cells treated with bicalutamide alone did not exhibit any reduction in cystine levels.…”

The lysosomal V-ATPase B1 subunit in renal proximal tubule is linked to nephropathic cystinosis

“…Cystine levels were quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS); a rapid and sensitive assay that has been developed and validated in house. 22 In brief, cell pellets were suspended in N-Ethylmaleimide (NEM) solution containing 5 mM NEM in 0.1 mM sodium phosphate buffer (pH 7.4). The cell suspension was then precipitated, and protein was extracted with sulfosalicylic acid 15% (w/v) and centrifuged at 20,000 g for 10 min at 4 o C. Protein concentration was determined by the method of the Pierce™ BCA protein assay kit according to the manufacturer's protocol (Thermo Fisher, The Netherlands), and the cystine concentration was measured using HPLC-MS/MS.…”