Zhibin Ning

Proteomic reactors and their applications in biology.

Proteomic analysis requires the combination of an extensive suite of technologies including protein processing and separation, micro‐flow HPLC, MS and bioinformatics. Although proteomic technologies are still in flux, approaches that bypass gel electrophoresis (gel‐free approaches) are dominating the field of proteomics. Along with the development of gel‐free proteomics, came the development of devices for the processing of proteomic samples termed proteomic reactors. These microfluidic devices provide rapid, robust and efficient pre‐MS sample procession by performing protein sample preparation/concentration, digestion and peptide fractionation. The proteomic reactor has advanced in two major directions: immobilized enzyme reactor and ion exchange‐based proteomic reactor. This review summarizes the technical developments and biological applications of the proteomic reactor over the last decade.

mChIP-KAT-MS, a method to map protein interactions and acetylation sites for lysine acetyltransferases

Significance Recent proteomic studies have revealed that lysine acetylation is a global and ubiquitous posttranslational modification. However, in the vast majority of cases the lysine acetyltransferases (KATs) responsible for individual modifications remain unknown. Here we present a unique methodology that connects KATs to their substrates. To validate the methodology, we use the yeast KAT nucleosome acetyltransferase of histone H4 (NuA4) and identify both protein interactions and acetylation targets. Importantly, this methodology can be applied to any KAT and should aid in the linking of KATs to their cellular targets. Recent global proteomic and genomic studies have determined that lysine acetylation is a highly abundant posttranslational modification. The next challenge is connecting lysine acetyltransferases (KATs) to their cellular targets. We hypothesize that proteins that physically interact with KATs may not only predict the cellular function of the KATs but may be acetylation targets. We have developed a mass spectrometry-based method that generates a KAT protein interaction network from which we simultaneously identify both in vivo acetylation sites and in vitro acetylation sites. This modified chromatin-immunopurification coupled to an in vitro KAT assay with mass spectrometry (mChIP-KAT-MS) was applied to the Saccharomyces cerevisiae KAT nucleosome acetyltransferase of histone H4 (NuA4). Using mChIP-KAT-MS, we define the NuA4 interactome and in vitro-enriched acetylome, identifying over 70 previously undescribed physical interaction partners for the complex and over 150 acetyl lysine residues, of which 108 are NuA4-specific in vitro sites. Through this method we determine NuA4 acetylation of its own subunit Epl1 is a means of self-regulation and identify a unique link between NuA4 and the spindle pole body. Our work demonstrates that this methodology may serve as a valuable tool in connecting KATs with their cellular targets.

Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor

Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.

The proteomic landscape of the suprachiasmatic nucleus clock reveals large-scale coordination of key biological processes.

The suprachiasmatic nucleus (SCN) acts as the central clock to coordinate circadian oscillations in mammalian behavior, physiology and gene expression. Despite our knowledge of the circadian transcriptome of the SCN, how it impacts genome-wide protein expression is not well understood. Here, we interrogated the murine SCN proteome across the circadian cycle using SILAC-based quantitative mass spectrometry. Of the 2112 proteins that were accurately quantified, 20% (421 proteins) displayed a time-of-day-dependent expression profile. Within this time-of-day proteome, 11% (48 proteins) were further defined as circadian based on a sinusoidal expression pattern with a ∼24 h period. Nine circadianly expressed proteins exhibited 24 h rhythms at the transcript level, with an average time lag that exceeded 8 h. A substantial proportion of the time-of-day proteome exhibited abrupt fluctuations at the anticipated light-to-dark and dark-to-light transitions, and was enriched for proteins involved in several key biological pathways, most notably, mitochondrial oxidative phosphorylation. Additionally, predicted targets of miR-133ab were enriched in specific hierarchical clusters and were inversely correlated with miR133ab expression in the SCN. These insights into the proteomic landscape of the SCN will facilitate a more integrative understanding of cellular control within the SCN clock.

Quantitative site-specific ADP-ribosylation profiling of DNA-dependent PARPs

An important feature of poly(ADP-ribose) polymerases (PARPs) is their ability to readily undergo automodification upon activation. Although a growing number of substrates were found to be poly(ADP-ribosyl)ated, including histones and several DNA damage response factors, PARPs themselves are still considered as the main acceptors of poly(ADP-ribose). By monitoring spectral counts of specific hydroxamic acid signatures generated after the conversion of the ADP-ribose modification onto peptides by hydroxylamine hydrolysis, we undertook a thorough mass spectrometry mapping of the glutamate and aspartate ADP-ribosylation sites onto automodified PARP-1, PARP-2 and PARP-3. Thousands of hydroxamic acid-conjugated peptides were identified with high confidence and ranked based on their spectral count. This semi-quantitative approach allowed us to locate the preferentially targeted residues in DNA-dependent PARPs. In contrast to what has been reported in the literature, automodification of PARP-1 is not predominantly targeted towards its BRCT domain. Our results show that interdomain linker regions that connect the BRCT to the WGR module and the WGR to the PRD domain undergo prominent ADP-ribosylation during PARP-1 automodification. We also found that PARP-1 efficiently automodifies the D-loop structure within its own catalytic fold. Interestingly, additional major ADP-ribosylation sites were identified in functional domains of PARP-1, including all three zinc fingers. Similar to PARP-1, specific residues located within the catalytic sites of PARP-2 and PARP-3 are major targets of automodification following their DNA-dependent activation. Together our results suggest that poly(ADP-ribosyl)ation hot spots make a dominant contribution to the overall automodification process.

From cells to peptides: "one-stop" integrated proteomic processing using amphipols.

In proteomics, detergents and chaotropes are indispensable for proteome analysis, not only for protein extraction, but also for protein digestion. To increase the protein extraction efficiency, detergents are usually added in the lysis buffer to extract membrane proteins out of membrane structure and to maintain protein in solutions. In general, these detergents need to be removed prior to protein digestion, usually by precipitation or ultrafiltration. Digestion often takes place in the presence of chaotropic reagents, such as urea, which often need to be removed prior to mass spectrometry. The addition and removal of detergents and chaotropes require multiple steps that are time-consuming and can cause sample losses. Amphipols (APols) are a different class of detergents that have physical and solubilization properties that are distinct from conventional detergents. They have primarily been used in protein structure analysis for membrane protein trapping and stabilization. Here, we demonstrate a simple and rapid protocol for total and membrane proteome preparation using APols. We demonstrate that APols added for cell lysis help maintain the proteome in solution, are compatible with protein digestion using trypsin, and can readily be removed prior to mass spectrometry by a one-step acidification and centrifugation. This protocol is much faster, can be performed in a single tube, and can readily replace the conventional detergent/chaotrope approaches for total and membrane proteome analysis.

A New Chemical Probe for Phosphatidylinositol Kinase Activity

Phosphatidylinositol kinases (PIKs) are key enzymatic regulators of membrane phospholipids and membrane environments that control many aspects of cellular function, from signal transduction to secretion, through the Golgi apparatus. Here, we have developed a photoreactive “clickable” probe, PIK‐BPyne, to report the activity of PIKs. We investigated the selectivity and efficiency of the probe to both inhibit and label PIKs, and we compared PIK‐BPyne to a wortmannin activity‐based probe also known to target PIKs. We found that PIK‐BPyne can act as an effective in situ activity‐based probe, and for the first time, report changes in PI4K‐IIIβ activity induced by the hepatitis C virus. These results establish the utility of PIK‐BPyne for activity‐based protein profiling studies of PIK function in native biological systems.

Discovery of Substrates for a SET Domain Lysine Methyltransferase Predicted by Multistate Computational Protein Design

Characterization of lysine methylation has proven challenging despite its importance in biological processes such as gene transcription, protein turnover, and cytoskeletal organization. In contrast to other key posttranslational modifications, current proteomics techniques have thus far shown limited success at characterizing methyl-lysine residues across the cellular landscape. To complement current biochemical characterization methods, we developed a multistate computational protein design procedure to probe the substrate specificity of the protein lysine methyltransferase SMYD2. Modeling of substrate-bound SMYD2 identified residues important for substrate recognition and predicted amino acids necessary for methylation. Peptide- and protein- based substrate libraries confirmed that SMYD2 activity is dictated by the motif [LFM]-1-K(∗)-[AFYMSHRK]+1-[LYK]+2 around the target lysine K(∗). Comprehensive motif-based searches and mutational analysis further established four additional substrates of SMYD2. Our methodology paves the way to systematically predict and validate posttranslational modification sites while simultaneously pairing them with their associated enzymes.

Methylmercury can induce Parkinson's-like neurotoxicity similar to 1-methyl-4- phenylpyridinium: a genomic and proteomic analysis on MN9D dopaminergic neuron cells.

Exposure to environmental chemicals has been implicated as a possible risk factor for the development of neurodegenerative diseases. Our previous study showed that methylmercury (MeHg) exposure can disrupt synthesis, uptake and metabolism of dopamine similar to 1-methyl-4-phenylpyridinium (MPP(+)). The objective of this study was to investigate the effects of MeHg exposure on gene and protein profiles in a dopaminergic MN9D cell line. MN9D cells were treated with MeHg (1-5 μM) and MPP(+) (10-40 μM) for 48 hr. Real-time PCR Parkinsons disease (PD) arrays and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) were performed for the analysis. PD PCR array results showed that 19% genes were significantly changed in the 2.5 μM MeHg treated cells, and 39% genes were changed in the 5 μM MeHg treated cells. In comparison, MPP(+) treatment (40 µM) resulted in significant changes in 25% genes. A total of 15 common genes were altered by both MeHg and MPP(+), and dopaminergic signaling transduction was the most affected pathway. Proteomic analysis identified a total of 2496 proteins, of which 188, 233 and 395 proteins were differentially changed by 1 μM and 2.5 μM MeHg, and MPP(+) respectively. A total of 61 common proteins were changed by both MeHg and MPP(+) treatment. The changed proteins were mainly involved in energetic generation-related metabolism pathway (propanoate metabolism, pyruvate metabolism and fatty acid metabolism), oxidative phosphorylation, proteasome, PD and other neurodegenerative disorders. A total of 7 genes/proteins including Ube2l3 (Ubiquitin-conjugating enzyme E2 L3) and Th (Tyrosine 3-monooxygenase) were changed in both genomic and proteomic analysis. These results suggest that MeHg and MPP(+) share many similar signaling pathways leading to the pathogenesis of PD and other neurodegenerative diseases.

Fine Tuning of Proteomic Technologies to Improve Biological Findings: Advancements in 2011–2013

■ CONTENTS Sample Preparation 177 Protein Extraction 177 Secreted Proteins 177 Exosomes 177 Membrane Proteins 178 Protein Stabilization 178 Miniturizing and Automating Sample Preparation 178 Protein Digestion 178 Multiple Enzymes 178 Enzyme Immobilization 178 Decreasing Sample Complexity 178 Serum/Plasma Strategies to Improve Dynamic Range 178 Depletion Strategies 178 Enrichment Strategies 179 Enriching Post-Translationally Modified Proteomes 180 Phosphopeptide Enrichment 180 Glycopeptide Enrichment 180 Enrichment of Other PTMs 181 Methods to Improve Coverage 181 Filtered Aided Sample Preparation (FASP) 181 SDS Spin Columns 181 Detergent Clean-up Methods for MS-Deleterious Agents 182 Integrated Approaches 182 Monolithic Columns 182 Quantitative Proteomics 182 Gel Staining 182 Label-Free Quantitation 182 Metabolic Labeling/SILAC 183 Chemical Labeling 183 Targeted Quantitative Proteomics 183 Selected Reaction Monitoring 183 Characterizing Post-Translationally Modified Proteomes by Mass Spectrometry 184 Protein Interactions 185 Technologies 185 Methods 185 Applications 186 Bioinformatics 186 Proteomic Analyses/Bioinformatics 187 Databases 187 Search Engines 187 Software 188 Label-Free Software 188 Extended Analysis 188 From Proteomic to Biological Applications 188 Profiling Disease States 188 From Genes to Proteins 188 Biomarker Development 189 Identification of Novel Biomarkers 189 Applying Proteomics to Known Biomarkers 189 Conclusions 190 Author Information 190 Corresponding Author 190 Notes 190 Biographies 190 Acknowledgments 190 References 190