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.

Figure 1. Sirtuin peptide standard curves, variance and limits of detection and quantification.

Sirtuin peptide standard curves (average of the peak area ratios for the two peptides, in triplicate for each sirtuin, with heavy peptide spike of 100 fmol/μl). Both inter- and intra-assay variance were calculated for three replicates and the LOD and LOQ are shown in fmol/ul. CVs calculated using peptide peak area ratios (light/heavy) at the 100 fmol/μl peptide concentration level. Individual chromatograms for each of the 14 peptides can be found in Supplementary Figure S1.

Figure 2. Sirtuin expression in primary cultured brain cells and cell lines.

In primary neurons SIRT1 was found to be the most abundant sirtuin (*p < 0.05) compared to other neuronal sirtuins (n = 3). SIRT2 was found to be abundant in primary oligodendrocytes (*p < 0.05) compared to other cell types (n = 3). SIRT1 and SIRT2 were the most abundant in all the cell cultures. SIRT4 and SIRT5 were below the detection limits and only small amounts of SIRT6 and SIRT7 were detected.

Figure 3. Sirtuin expression in human frontal lobe brain tissue.

Sirtuin expression in unfractionated (Panel A) and fractionated (Panel B) human frontal lobe brain tissue. All seven sirtuins were detected in unfractionated human frontal lobe brain tissue with SIRT2 the most abundant (*p < 0.05) compared to all other sirtuins (Panel A). The mitochondrial sirtuins (SIRT3-5) were found to be close to the LOQ, but their signals improved following fractionation of the tissue into cytosolic, nuclear, cytoskeletal and membrane fractions (Panel B). Fractionation showed SIRT1 and SIRT2 expressed in all fractions, whereas SIRT3-5 were below the LOD in the cytosol and SIRT6 and SIRT7 were below the LOD in the cytoskeletal fraction. SIRT1 was most abundant in the nucleus and SIRT2 in the cytoskeletal fraction (*p < 0.05, Panel B). In the cytosol, SIRT2 was the most abundant sirtuin (*p < 0.05), and all sirtuins were captured in the membrane fraction.

Figure 4. SIRT1-3 protein expression in animal organs.

SIRT1-3 protein expression in guinea pig (Panel A) and mouse organs (Panel B). SIRT2 was found to be the most abundant sirtuin in guinea pig, with higher levels expressed in the brain (*p < 0.01, n = 2) compared to liver and kidney. SIRT2 in mouse was not quantified due to peptide sequence difference with the human peptide standards.

Figure 5

Sirtuin expression in human brain cells and tissue using immunohistochemical staining of astrocytes (Panel A row 1–4) and human frontal lobe brain tissue (Panel A, row 5), PCR of SIRT1 and SIRT2 mRNA in occipital, frontal and hippocampal human control brain (Panel B) and cropped images of western blotting of SIRT1-3 protein expression in human control frontal lobe brain tissue (n = 3) at molecular weights of approx 40 kDa, 50 kDa and 30 kDa respectively (Panel C). Full length blots are presented in Supplementary Figure S4.