Label-Free Mass Spectrometry-Based Quantitative Proteomics to Evaluate the Effects of the Calcium-Sensing Receptor Agonist Cinacalcet on Protein Expression in Rat Brains and Livers

Abstract

Background Cinacalcet is a calcium-sensing receptor agonist that is clinically approved for the treatment of secondary hyperparathyroidism in chronic kidney disease and hypercalcemia in patients with parathyroid carcinoma. This study aimed to use quantitative mass spectrometry-based label-free proteomics to evaluate the effects of cinacalcet on protein expression in rat brains and livers. Material/Methods We randomly assigned 18 Wistar rats to 2 groups: an untreated control group (n=6) and a group treated with cinacalcet at a dose corresponding to the maximum dose used in humans (2 mg/kg/body weight, 5 days/week) divided into 7-day (n=6) and 21-day (n=6) treatment subgroups. A mass-spectrometry-based label-free quantitative proteomics approach using peptides peak area calculation was used to evaluate the changes in protein expression in examined tissues. Bioinformatics analysis of quantitative proteomics data was done using MaxQuant and Perseus environment. Results No changes in protein expression were revealed in the 7-day treatment subgroup. We detected 10 upregulated and 3 downregulated proteins in the liver and 1 upregulated protein in the brain in the 21-day treatment subgroup compared to the control group. Based on Gene Ontology classification, all identified differentially expressed proteins were indicated as molecular functions involved in the enzyme regulator activity (36%), binding (31%), and catalytic activity (19%). Conclusions These findings indicate that long-term cinacalcet therapy can impair phase II of enzymatic detoxication and can cause disturbances in blood hemostasis, lipid metabolism, and inflammatory mediators or contribute to the acceleration of cognitive dysfunction; therefore, appropriate patient monitoring should be considered.

A Standardized Protocol for Assuring the Validity of Proteomics Results from Liquid Chromatography–High-Resolution Mass Spectrometry

“…Our experience demonstrates that using a single protein (BSA) as a control sample in many complex analyses might not be sufficient. This is a major problem in quantitative proteomic studies without the use of isotopic labeling [ 5 , 6 ], where direct information about protein concentration is obtained from the intensity of the chromatographic signal. In such cases, it is necessary to ensure that each of the several chromatographic runs in the measurement series was performed under the same conditions.…”

“…The approach proposed in our latest papers [ 5 , 6 ] is also an interesting concept for preparing a relevant complex control sample for proteomics studies. In the case of quantitative analysis, a control sample was prepared for testing the stability of the measurement system by mixing an appropriate portion of all samples from the measurement series into a single complex control sample ( Figure 2 ).…”

“…In a wider meaning, proteomics is the study of the interactions, function, composition, and structures of proteins and their cellular activities [ 2 ]. Mass spectrometry-based proteomics has contributed significantly to the detection of drug target identification [ 3 ], and alteration of expression patterns in response to different environmental factors such as diet enriched in selenium compounds [ 4 , 5 ] or fluorinated drugs administration [ 6 , 7 ]. Modern proteomics methods have also recently become useful in neurodegenerative diseases in developing methods for the analysis [ 8 ] and identification of new biomarkers for early diagnosis and disease progression [ 9 , 10 , 11 , 12 ].…”

“…Proteomic analysis, using high-resolution mass spectrometry, is a multistage process involving: (i) sample preparation for measurement; (ii) chromatographic separation of a mixture of proteins or peptides and analysis of the specific components of the sample using an appropriate MS analyzer; and (iii) interpretation of the results obtained using relevant databases and bioinformatic software tools. The advanced techniques based on high-resolution mass spectrometry are characterized by high complexity, and mass spectrometry-based proteomics experiments might be affected by the variation in individual samples in a measurement series over time, especially in quantitative methods [ 5 , 6 , 22 , 23 , 24 , 25 , 26 ], which is a limitation in obtaining accurate and reproducible results. Papers on proteomic analysis often omit a clear description of how the quality of the results was ensured and how their validity was evaluated.…”