Application of Targeted Mass Spectrometry for the Quantification of Sirtuins in the Central Nervous System

Abstract

Sirtuin proteins have a variety of intracellular targets, thereby regulating multiple biological pathways including neurodegeneration. However, relatively little is currently known about the role or expression of the 7 mammalian sirtuins in the central nervous system. Western blotting, PCR and ELISA are the main techniques currently used to measure sirtuin levels. To achieve sufficient sensitivity and selectivity in a multiplex-format, a targeted mass spectrometric assay was developed and validated for the quantification of all seven mammalian sirtuins (SIRT1-7). Quantification of all peptides was by multiple reaction monitoring (MRM) using three mass transitions per protein-specific peptide, two specific peptides for each sirtuin and a stable isotope labelled internal standard. The assay was applied to a variety of samples including cultured brain cells, mammalian brain tissue, CSF and plasma. All sirtuin peptides were detected in the human brain, with SIRT2 being the most abundant. Sirtuins were also detected in human CSF and plasma, and guinea pig and mouse tissues. In conclusion, we have successfully applied MRM mass spectrometry for the detection and quantification of sirtuin proteins in the central nervous system, paving the way for more quantitative and functional studies.

RTN4B‐mediated suppression of Sirtuin 2 activity ameliorates β‐amyloid pathology and cognitive impairment in Alzheimer's disease mouse model

“…The sirtuin family of proteins, which includes seven mammalian members, are famous protein deacetylases that participate in considerable biological and pathological processes, highlighting their crucial physiological functions (Finkel, Deng, & Mostoslavsky, 2009; Gomes, Leal, Mendes, Reis, & Cavadas, 2019; Lin et al, 2018). Among them, SIRT2 is the only sirtuin mainly located in the cytoplasm and abundantly expressed in the brain (Jayasena et al, 2016). Moreover, SIRT2 also accumulates in the aging central nervous system (CNS), marking its potential role in aging or related neurological diseases (Maxwell et al, 2011).…”

Cerebellar Predominant Increase in mRNA Expression Levels of Sirt1 and Sirt3 Isoforms in a Transgenic Mouse Model of Huntington’s Disease

“…Western blotting) are semiquantitative at best, mostly lacking appropriate sensitivity and specificity [29]. Although the application of deep proteomic analysis by mass spectrometry is appropriate, but quite challenging [21], especially in case of brain samples with considerably high lipid content. Accordingly, this was out of the scope of the current study.…”

Role of Sirtuins in Modulating Neurodegeneration of the Enteric Nervous System and Central Nervous System

“…All of the seven sirtuins are ubiquitously expressed in all human tissues (Yamamoto et al, 2007 ). Mass spectrometry and semi-quantitative studies have shown that all the sirtuins are expressed in the human and non-human brain and small intestine (Sidorova-Darmos et al, 2014 ; Jayasena et al, 2016 ). In the brain, SIRT1 (110 KDa) and SIRT2 (37 KDa) are the most abundant and widely expressed sirtuin subtypes.…”