Sirtuins: Methods and Protocols

Part I: Methods to Generate Sirtuin Biology Tools

1. Introduction: Sirtuins in Aging and Diseases

Leonard Guarente

2. Sirtuins in Yeast: Phenotypes and Tools

Scott Tsuchiyama, Elizabeth Kwan, Weiwei Dang, Antonio Bedalov, and Brian K. Kennedy

3. Studying the C. elegans Sirtuins

Mohan Viswanathan and Heidi A. Tissenbaum

4. Genetic and Biochemical Tools for Investigating Sirtuin Function in Drosophila melanogaster

Jason G. Wood, Rachel Whitaker, and Stephen L. Helfand

5. Generating Mammalian Sirtuin Tools for Protein-Interaction Analysis

Kathleen A. Hershberger, Jonathan Motley, Matthew D. Hirschey, and Kristin A. Anderson

Part II: Methods to Identify Sirtuin Substrates

6. Mass Spectrometry-Based Detection of Protein Acetylation

Yu Li, Jeffrey C. Silva, Mary E. Skinner, and David B. Lombard

7. SILAC-Based Quantification of Sirt1-Responsive Lysine Acetylome

Yue Chen, Gozde Colak, and Yingming Zhao

8. Targeted Quantitation of Acetylated Lysine Peptides by Selected Reaction Monitoring Mass Spectrometry

Matthew J. Rardin, Jason M. Held, and Bradford W. Gibson

9. Identification of Deacetylase Substrates with the Biotin Switch Approach

J. Will Thompson, Alex Robeson, and Joshua L. Andersen

10. Assaying Chromatin Sirtuins

Lei Zhong, Barbara Martinez-Pastor, Dafne M. Silberman, Carlos Sebastian, and Raul Mostoslavsky

Part III: Methods to Measure Sirtuin Activity

11. Measurement of Sirtuin Enzyme Activity Using a Substrate-Agnostic Fluorometric Nicotinamide Assay

Basil P. Hubbard and David A. Sinclair

12. Detecting Sirtuin-Catalyzed Deacylation Reactions Using ³²P-Labeled NAD and Thin-Layer Chromatography

Anita Zhu, Xiaoyang Su, and Hening Lin

13. Chemical Acetylation And Deacetylation

Kristofer S. Fritz

14. Accurate Measurement of Nicotinamide Adenine Dinucleotide (NAD⁺) with High-Performance Liquid Chromatography

Jun Yoshino and Shin-ichiro Imai

15. In Vivo Measurement of the Acetylation State of Sirtuin Substrates as a Proxy for Sirtuin Activity

John Dominy, Pere Puigserver, and Carles Cantó

Part IV: Methods to Study Sirtuin Biology

16. Oxygen Flux Analysis to Understand the Biological Function of Sirtuins

Dongning Wang, Michelle F. Green, Eoin McDonnell, and Matthew D. Hirschey

17. The Emerging Links Between Sirtuins and Autophagy

In Hye Lee, Jeanho Yun, and Toren Finkel

18. Methods to Study the Role of Sirtuins in Genome Stability

Paloma Martínez-Redondo and Alejandro Vaquero

19. Circadian Measurements of Sirtuin Biology

Kathryn Moynihan Ramsey, Alison H. Affinati, Clara B. Peek, Biliana Marcheva, Hee-Kyung Hong, and Joseph Bass

20. Utilizing Calorie Restriction to Evaluate the Role of Sirtuins in Healthspan and Lifespan of Mice

Jessica Curtis and Rafael de Cabo