James A. Dowell

Comparison of Two-Dimensional Fractionation Techniques for Shotgun Proteomics

Two-dimensional (2D) fractionation is a commonly used tool to increase dynamic range and proteome coverage for bottom-up, shotgun proteomics. However, there are few reports comparing the relative separation efficiencies of 2D methodologies using low-microgram sample quantities. In order to systematically evaluate 2D separation techniques, we fractionated microgram quantities of E. coli protein extract by seven different methods. The first dimension of separation was performed with either reversed-phase high-pressure liquid chromatography (RP-HPLC), gel electrophoresis (SDS-PAGE), or strong cation exchange (SCX-HPLC). The second dimension consisted of a standard reversed-phase capillary HPLC coupled to an electrospray ionization quadrupole time-of-flight mass spectrometer for tandem mass spectrometric analysis. The overall performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The protein-level RP-HPLC and the high-pH RP-HPLC peptide-level separations performed the best, identifying 281 and 266 proteins, respectively. The online pH variance SCX and the SDS-PAGE returned modest performances with 178 and 139 proteins identified, respectively. The offline SCX had the worst performance with 81 proteins identified. We also examined various chromatographic factors that contribute to separation efficiency, including resolving power, orthogonality, and sample loss.

Stoichiometry of site-specific lysine acetylation in an entire proteome.

Background: Lysine acetylation sites have been mapped, but information on stoichiometry is lagging. Results: We developed and utilized the first direct, unbiased method for quantifying site-specific acetylation stoichiometry of a proteome without antibody enrichment. Conclusion: High stoichiometry is associated with central metabolism, transcription, and translation. Loss of deacetylase CobB affects site-specific and global acetylation stoichiometry, altering acetyl-CoA metabolism. Significance: Stoichiometry provides functional insight into protein acetylation. Acetylation of lysine ϵ-amino groups influences many cellular processes and has been mapped to thousands of sites across many organisms. Stoichiometric information of acetylation is essential to accurately interpret biological significance. Here, we developed and employed a novel method for directly quantifying stoichiometry of site-specific acetylation in the entire proteome of Escherichia coli. By coupling isotopic labeling and a novel pairing algorithm, our approach performs an in silico enrichment of acetyl peptides, circumventing the need for immunoenrichment. We investigated the function of the sole NAD+-dependent protein deacetylase, CobB, on both site-specific and global acetylation. We quantified 2206 peptides from 899 proteins and observed a wide distribution of acetyl stoichiometry, ranging from less than 1% up to 98%. Bioinformatic analysis revealed that metabolic enzymes, which either utilize or generate acetyl-CoA, and proteins involved in transcriptional and translational processes displayed the highest degree of acetylation. Loss of CobB led to increased global acetylation at low stoichiometry sites and induced site-specific changes at high stoichiometry sites, and biochemical analysis revealed altered acetyl-CoA metabolism. Thus, this study demonstrates that sirtuin deacetylase deficiency leads to both site-specific and global changes in protein acetylation stoichiometry, affecting central metabolism.

Identification of Astrocyte Secreted Proteins with a Combination of Shotgun Proteomics and Bioinformatics

Astrocytes are important regulators of normal brain function in mammals, including roles in synaptic signaling, synapse formation, and neuronal health and survival. Many of these functions are executed via secreted proteins. To analyze the astrocyte secretome, a combination of shotgun proteomics and bioinformatics was employed to analyze conditioned media from primary murine astrocyte cultures. Both two- and one-dimensional LC-MS/MS were used to analyze astrocyte secreted proteins, resulting in the identification of over 420 proteins. To refine our results, the intracellular protein contaminants were removed in silico using a cytoplasmic control. In additional rounds of refinement, putative secreted proteins were subjected to analysis by SignalP, SecretomeP, and gene ontology analysis, yielding a refined list of 187 secreted proteins. In conclusion, the use of shotgun proteomics combined with multiple rounds of data refinement produced a high quality catalog of astrocyte secreted proteins.

Quantification of SAHA-Dependent Changes in Histone Modifications Using Data-Independent Acquisition Mass Spectrometry

Histone post-translational modifications (PTMs) are important regulators of chromatin structure and gene expression. Quantitative analysis of histone PTMs by mass spectrometry remains extremely challenging due to the complex and combinatorial nature of histone PTMs. The most commonly used mass spectrometry-based method for high-throughput histone PTM analysis is data-dependent acquisition (DDA). However, stochastic precursor selection and dependence on MS1 ions for quantification impede comprehensive interrogation of histone PTM states using DDA methods. To overcome these limitations, we utilized a data-independent acquisition (DIA) workflow that provides superior run-to-run consistency and postacquisition flexibility in comparison to DDA methods. In addition, we developed a novel DIA-based methodology to quantify isobaric, co-eluting histone peptides that lack unique MS2 transitions. Our method enabled deconvolution and quantification of histone PTMs that are otherwise refractory to quantitation, including the heavily acetylated tail of histone H4. Using this workflow, we investigated the effects of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) on the global histone PTM state of human breast cancer MCF7 cells. A total of 62 unique histone PTMs were quantified, revealing novel SAHA-induced changes in acetylation and methylation of histones H3 and H4.

Mechanisms of Nrf2 Protection in Astrocytes as Identified by Quantitative Proteomics and siRNA Screening

The Nrf2 (NF-E2 related factor 2)-ARE (antioxidant response element) pathway controls a powerful array of endogenous cellular antioxidant systems and is an important pathway in the detoxification of reactive oxygen species (ROS) in the brain. Using a combination of quantitative proteomics and siRNA screening, we have identified novel protective mechanisms of the Nrf2-ARE pathway against oxidative stress in astrocytes. Studies from our lab and others have shown Nrf2 overexpression protects astrocytes from oxidative stress. However, the exact mechanisms by which Nrf2 elicits these effects are unknown. In this study, we show that induction of Nrf2 reduces levels of reactive oxygen species (ROS) produced by various oxidative stressors and results in robust cytoprotection. To identify the enzymes responsible for these effects, we used stable isotope labeling by amino acids in cell culture (SILAC) and quantitative shotgun proteomics to identify 72 Nrf2-regulated proteins in astrocytes. We hypothesized a subset of these proteins might play a critical role in Nrf2 protection. In order to identify these critical proteins, we used bioinformatics to narrow our target list of proteins and then systematically screened each candidate with siRNA to assess the role of each in Nrf2 protection. We screened each target against H2O2, tert-butyl hydroperoxide, and 4-hydroxynonenal and subsequently identified three enzymes–catalase, prostaglandin reductase-1, and peroxiredoxin-6–that are critical for Nrf2-mediated protection in astrocytes.

Iron Deprivation Induces Transcriptional Regulation of Mitochondrial Biogenesis.

Mitochondria are essential organelles that adapt to stress and environmental changes. Among the nutrient signals that affect mitochondrial form and function is iron, whose depletion initiates a rapid and reversible decrease in mitochondrial biogenesis through unclear means. Here we demonstrate that, unlike the canonical iron-induced alterations to transcript stability, loss of iron dampens the transcription of genes encoding mitochondrial proteins with no change to transcript half-life. Using mass spectrometry, we demonstrate that these transcriptional changes are accompanied by dynamic alterations to histone acetylation and methylation levels that are largely reversible upon readministration of iron. Moreover, histone deacetylase inhibition abrogates the decreased histone acetylation observed upon iron deprivation and restores normal transcript levels at genes encoding mitochondrial proteins. Collectively, we demonstrate that deprivation of an essential nutrient induces transcriptional repression of organellar biogenesis involving epigenetic alterations.

Rat Brain Neuropeptidomics: Tissue Collection, Protease Inhibition, Neuropeptide Extraction, and Mass Spectrometric Analysis

Due to the complexity of the mammalian central nervous system, neuropeptidomic studies in mammals often yield very complicated mass spectra that make data analysis difficult. Careful sample preparation and extraction protocols must be employed in order to minimize spectral complexity and enable extraction of useful information on neuropeptides from a given sample. Controlling post-mortem protease activity is essential to simplifying mass spectra and to identifying low-abundance neuropeptides in tissue samples. Post-mortem microwave-irradiation coupled with cryostat dissection has proven to be effective in arresting protease activity to allow detection of endogenous neuropeptides instead of protein degradation products.

Increased expression of AT-1/SLC33A1 causes an autistic-like phenotype in mice by affecting dendritic branching and spine formation

Increased expression of the ER membrane acetyl-CoA transporter AT-1 can cause an autism-like phenotype in mice.

Histone Lysine-to-Methionine Mutations Reduce Histone Methylation and Cause Developmental Pleiotropy

Transgenic plants expressing H3K36M mutations have a dominant-negative effect on the endogenous histone methylation landscape, leading to pleiotropic developmental defects. Epigenetic modifications play critical roles in diverse biological processes. Histone Lys-to-Met (K-to-M) mutations act as gain-of-function mutations to inhibit a wide range of histone methyltransferases and are thought to promote tumorigenesis. However, it is largely unknown whether K-to-M mutations impact organismal development. Using Arabidopsis (Arabidopsis thaliana) as a model system, we discovered that a transgene exogenously expressing histone 3 Lys-36 to Met mutation (K36M) acts in a dominant-negative manner to cause global reduction of H3K36 methylation. Remarkably, this dominant repressive activity is dosage-dependent and causes strong developmental perturbations including extreme branching and early flowering by affecting the expression of genes involved in developmental and metabolic processes. Besides the established pathological roles of K-to-M mutations in tumor cells, we demonstrate a physiological outcome for K-to-M induced H3K36 hypomethylation. This study provides evidence for a conserved dominant-negative inhibitory role of histone K-to-M mutation across the plant and animal kingdoms. We also highlight the unique ability of K36M mutations to alter plant developmental processes leading to severe pleiotropic phenotypes. Finally, our data suggests K-to-M mutations may provide a useful strategy for altering epigenetic landscapes in organisms where histone methyltransferases are uncharacterized.

c-Jun N-terminal kinase regulates mGluR-dependent expression of post-synaptic FMRP target proteins

Fragile X syndrome (FXS) is caused by the loss of functional fragile X mental retardation protein (FMRP). Loss of FMRP results in an elevated basal protein expression profile of FMRP targeted mRNAs, a loss of local metabotropic glutamate receptor (mGluR)‐regulated protein synthesis, exaggerated long‐term depression and corresponding learning and behavioral deficits. Evidence shows that blocking mGluR signaling in FXS models ameliorates these deficits. Therefore, understanding the signaling mechanisms downstream of mGluR stimulation may provide additional therapeutic targets for FXS. Kinase cascades are an integral mechanism regulating mGluR‐dependent protein translation. The c‐Jun N‐terminal kinase (JNK) pathway has been shown to regulate mGluR‐dependent nuclear transcription; however, the involvement of JNK in local, synaptic signaling has not been explored. Here, we show that JNK is both necessary and sufficient for mGluR‐dependent expression of a subset of FMRP target proteins. In addition, JNK activity is basally elevated in fmr1 knockout mouse synapses, and blocking JNK activity reduces the over‐expression of post‐synaptic proteins in these mice. Together, these data suggest that JNK may be an important signaling mechanism downstream of mGluR stimulation, regulating FMRP‐dependent protein synthesis. Furthermore, local, post‐synaptic dysregulation of JNK activity may provide a viable target to ameliorate the deficits involved in FXS.