The function and physical properties of proteins can be regulated by protein post-translational modifications (PTMs). Lysine acetylation is a reversible PTM that affects a variety of biological processes, such as protein-DNA interactions, enzyme activation/inactivation, subcellular localization and protein stability, etc 1, 2
. In the past four decades, histones have been the primary focus of acetylation studies because of their high abundance and high frequency of lysine acetylation. Only studies of specific acetylation in individual proteins were possible in the past due to the lack of robust detection technologies3–5
. Improvements of modern mass spectrometers, specifically improved accurate mass detection, sensitivity and dynamic range, higher resolution, and faster scan rates, all serve to greatly facilitate global acetylation studies6
as has been reported recently7–9
. In 2006, Kim et al.
published the first proteome-wide report by using immunoprecipitation enrichment of acetylated peptides with an anti-acetylated lysine antibody and HPLC-MSMS detection8
to identify 195 acetylated proteins from mouse liver. Choudhary et al
. and Zhao et al
. advanced this approach and demonstrated the largest data set of acetylated peptides identified from human cells (1750 acetylated proteins) and liver tissue (1047 acetylated proteins)7, 9
. These reports show large numbers of acetylated non-histone proteins, many of which are mitochondrial or cytoplasmic species. Thus, lysine acetylation is involved in a greater diversity of functional roles and subcellular localizations than previously recognized.
As lysine acetylation is dynamic and regulates protein function in ways not fully understood, global quantitative analyses of lysine acetylation are critical to better understand the functional impact and use of this modification in biological systems. For example, Kendrick et al
. showed fatty liver was associated with reduced sirtuin activity and increased acetylation levels of mitochondrial proteins10
. They isolated acetylated proteins from total liver proteome samples from mice on a high fat diet (HFD, 4 months) and control animals. Acetylated proteins were immunoprecipitated with immobilized anti-acetylated lysine antibodies, purified proteins were separated on 1-D gels, scanned, and relative quantification between HFD-fed and control animal samples was performed. Bands that exhibited significant differential staining between HFD and control samples were subjected to in-gel digestion and mass spectrometry analysis. In total, 193 proteins were differentially acetylated in HFD samples, including proteins involved in gluconeogenesis, mitochondrial protein oxidation, methionine metabolism, liver injury and endoplasmic reticulum stress response. Importantly, the work of Kendrick et al.
shows that differences in levels of acetylated liver proteins relevant to altered feeding status in mice can be observed by proteomic methods. Previously, Choudhary et al.
used SILAC (Stable isotope labeling with amino acids in cell culture)7
and Zhao et al.
used iTRAQ labeling (isobaric tag for relative and absolute quantification)9
coupled with LC-MSMS techniques to quantify peptide-level, or site-specific acetylation in human cell lines and liver tissue respectively. Schwer and co-workers11
reported label free quantification (LFQ) of the acetylated peptides from mouse liver tissue. LFQ technology is beneficial for studies of live animal tissues because incorporation of heavy isotopes is not required. In this way, Schwer et al
. studied how calorie restriction (CR) can alter the mitochondrial protein acetylation levels in mouse liver. Approximately 300 proteins were quantified, and 72 were determined to have at least a 2.5 fold change in acetylation during CR. SILAM (Stable Isotope Labeling in Mammals)12
can in principle allow isotope incorporation in these studies with live animal tissues, although the additional expense significantly limits the applicability. In this work, a further advance of these approaches includes measurement of differences of acetylation on specific peptides by quantitative LC-MSMS methods, and comparison of level changes observed among several tissues or organs.
Acetylation is dynamic and cellular acetylation status is dependent on the activities of acetylases and deacetylases. Intracellular acetyl CoA, used by acetylases, sits on the metabolic crossroads of glycolysis, fatty acid oxidation, ketogenesis, amino acid metabolism, and TCA cycle utilization for ATP synthesis, making acetyl CoA an ideal quantity for sensing (via the non-nuclear acetylome) metabolic network function. Intracellular NAD+
/NADH ratio, a central determinant of nutritional status also critically regulates the activity of NAD+
deacetylases. Metabolic inflexibility, or the inability of an organism to adapt and modify fuel oxidation in response to changes in nutrient availability, is characteristic of dysfunction seen in metabolic syndrome and Type II Diabetes13–18
. The hypothesis for this study is that fasted/re-fed characterization of the organ specific mitochondrial and cytoplasmic acetylome may yield insights into both normal functional roles of tissue specific fuel switching, as well as abnormalities seen in situations, like diabetes mellitus, where metabolic inflexibility is evident. Therefore, analyzing the acetylation pattern of major mammalian tissues that greatly differ metabolically and play a diverse role in energy homeostasis under different nutritional status would be highly informative. Previous studies have reported murine fasted and re-fed hepatic acetylome only8
. Our efforts in this study focus on the characterization of the fasted/re-fed acetylome patterns of tissues that are known to switch fuels between the fasted/fed states (liver, skeletal muscle, heart muscle, white adipose and brown adipose) or have a high metabolic rate relative to their mass (brain and kidney)19
. No one organ is responsible for the metabolic rate, but some organs (brain, kidney, heart, and gastrointestinal tract) contribute a much larger fraction of their metabolic rate than their fractional mass or volume of the body, whereas others (bone, white adipose tissue, skin, and skeletal muscle) contribute much less19
. Brown adipose tissue contributes to the metabolic rate in neonatal mammals, and small mammals adapted to cold environments21
. Metabolic consequences of the presence or absence of the thermogenic capacity of brown adipose tissue in mice has been reported21, 22
, so brown adipose was also used for this study; white adipose tissue was included for comparison. The metabolic rate may fall 10% during sleep, a normal fasted condition, and may fall by 40% during long-term starvation in humans20
, supporting the relevance of acetylome changes on energy metabolism found for metabolic network enzymes during dietary shifts.
In this study, the acetylation levels of peptides from mitochondrial proteins in each tissue under differential feeding status were of primary interest. In total, 733 acetylated peptides from 337 proteins were identified, out of which the levels of 58 acetylated peptides changed 3-fold or greater under fasted/re-fed conditions. These peptides constitute the top 5% largest changes in acetylation levels among the 733 acetylated peptides, based on two standard deviations from the center of the observed log10 (re-fed/fasted) distribution as a cut off. Thirty-one acetylated peptides of these 58 are from metabolic proteins or chaperones. Many of these protein acetylation events may be relevant metabolic changes that accompany fuel switching and will serve as targets for future investigation. Our results show that acetylation levels in ATP-generating or utilizing metabolic processes such as glycolysis, Krebs cycle, gluconeogenesis, lipid synthesis and oxidation likely serve as control points or play a role in modulation of these pathways. In fact, acetylation is a classic candidate for such critical regulation, owing to its requirement of acetyl CoA, an important intermediate molecule linking such major pathways, as its acetyl group donor. The results presented here are first to demonstrate that there variation exists in the global quantitative profiles of acetylated metabolic proteins in all these organs, that are dramatically affected by comparison of the fasted to re-fed state.