title: 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.
authors.affiliation: Centre for Healthy Brain Ageing (CHeBA), School of Psychiatry, University of New South Wales, Sydney, Australia.
authors.forename: T
authors.initials: T
authors.lastname: Jayasena
authors.affiliation: Centre for Healthy Brain Ageing (CHeBA), School of Psychiatry, University of New South Wales, Sydney, Australia.|Bioanalytical Mass Spectrometry Facility, Mark Wainwright Analytical Centre, University of New South Wales, Sydney, Australia.|School of Medical Sciences, University of New South Wales, Sydney, Australia.
authors.forename: A
authors.initials: A
authors.lastname: Poljak
authors.affiliation: Centre for Healthy Brain Ageing (CHeBA), School of Psychiatry, University of New South Wales, Sydney, Australia.
authors.forename: N
authors.initials: N
authors.lastname: Braidy
authors.affiliation: Bioanalytical Mass Spectrometry Facility, Mark Wainwright Analytical Centre, University of New South Wales, Sydney, Australia.
authors.forename: L
authors.initials: L
authors.lastname: Zhong
authors.affiliation: School of Medical Sciences, University of New South Wales, Sydney, Australia.
authors.forename: B
authors.initials: B
authors.lastname: Rowlands
authors.affiliation: Centre for Healthy Brain Ageing (CHeBA), School of Psychiatry, University of New South Wales, Sydney, Australia.
authors.forename: J
authors.initials: J
authors.lastname: Muenchhoff
authors.affiliation: School of Medical Sciences, University of New South Wales, Sydney, Australia.|Sydney Medical School, University of Sydney, Sydney, Australia.
authors.forename: R
authors.initials: R
authors.lastname: Grant
authors.affiliation: School of Medical Sciences, University of New South Wales, Sydney, Australia.
authors.forename: G
authors.initials: G
authors.lastname: Smythe
authors.affiliation: Minimally Invasive Cancer Centre, Prince of Wales Hospital, Sydney, Australia.
authors.forename: C
authors.initials: C
authors.lastname: Teo
authors.affiliation: Bioanalytical Mass Spectrometry Facility, Mark Wainwright Analytical Centre, University of New South Wales, Sydney, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Raftery
authors.affiliation: Centre for Healthy Brain Ageing (CHeBA), School of Psychiatry, University of New South Wales, Sydney, Australia.|Neuropsychiatric Institute, the Prince of Wales Hospital, Sydney, Australia.
authors.forename: P
authors.initials: P
authors.lastname: Sachdev
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000592:standards*; D002490:Central Nervous System / Q000737:chemistry / Q000201:enzymology*; D015870:Gene Expression; D006168:Guinea Pigs; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000201:enzymology; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D009206:Myocardium / Q000737:chemistry / Q000201:enzymology; D058953:Neural Stem Cells / Q000166:cytology / Q000201:enzymology; D011110:Polymorphism, Genetic; D061251:Primary Cell Culture; D037761:Sirtuins / Q000097:blood / Q000134:cerebrospinal fluid / Q000145:classification / Q000235:genetics*
chemical_list: D037761:Sirtuins