Yueju Wang

Improving the comprehensiveness and sensitivity of sheathless capillary electrophoresis-tandem mass spectrometry for proteomic analysis.

We describe a solid phase microextraction (SPME), multistep elution, transient isotachophoresis (tITP) capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) procedure which employs a high sensitivity porous electrospray ionization (ESI) sprayer for the proteomic analysis of a moderately complex protein mixture. In order to improve comprehensiveness and sensitivity over a previously reported proteomic application of the ESI sprayer, we evaluated preconcentration with SPME and multistep elution prior to tITP stacking and CE separation. To maximize separation efficiency, we primarily employed electrokinetic methods for elution and separation after loading the sample by application of pressure. Conditions were developed for optimum simultaneous electrokinetic elution and sample stacking using a tryptic digest of 16 proteins to maximize peptide identifications and minimize band broadening. We performed comparative proteomic analysis of a dilution series using CE and nanoflow liquid chromatography (nLC). We found complementary peptide and protein identifications with larger quantities (100 ng) of a Pyrococcus furiosus tryptic digest, but with mass-limited amounts (5 ng) CE was 3 times more effective at identifying proteins. We attribute these gains in sensitivity to lower noise levels with the porous CE sprayer, illustrated by better signal-to-noise ratios of peptide precursor ions and associated higher XCorr values of identified peptides when compared directly to nLC. From comparative analysis of SPME-tITP-CE with direct injection CE, the SPME-tITP process improved comprehensiveness and sensitivity.

Systematic characterization of the murine mitochondrial proteome using functionally validated cardiac mitochondria

Mitochondria play essential roles in cardiac pathophysiology and the murine model has been extensively used to investigate cardiovascular diseases. In the present study, we characterized murine cardiac mitochondria using an LC/MS/MS approach. We extracted and purified cardiac mitochondria; validated their functionality to ensure the final preparation contains necessary components to sustain their normal function; and subjected these validated organelles to LC/MS/MS‐based protein identification. A total of 940 distinct proteins were identified from murine cardiac mitochondria, among which, 480 proteins were not previously identified by major proteomic profiling studies. The 940 proteins consist of functional clusters known to support oxidative phosphorylation, metabolism, and biogenesis. In addition, there are several other clusters, including proteolysis, protein folding, and reduction/oxidation signaling, which ostensibly represent previously under‐appreciated tasks of cardiac mitochondria. Moreover, many identified proteins were found to occupy other subcellular locations, including cytoplasm, ER, and golgi, in addition to their presence in the mitochondria. These results provide a comprehensive picture of the murine cardiac mitochondrial proteome and underscore tissue‐ and species‐specification. Moreover, the use of functionally intact mitochondria insures that the proteomic observations in this organelle are relevant to its normal biology and facilitates decoding the interplay between mitochondria and other organelles.

Phosphoproteome analysis reveals regulatory sites in major pathways of cardiac mitochondria

Mitochondrial functions are dynamically regulated in the heart. In particular, protein phosphorylation has been shown to be a key mechanism modulating mitochondrial function in diverse cardiovascular phenotypes. However, site-specific phosphorylation information remains scarce for this organ. Accordingly, we performed a comprehensive characterization of murine cardiac mitochondrial phosphoproteome in the context of mitochondrial functional pathways. A platform using the complementary fragmentation technologies of collision-induced dissociation (CID) and electron transfer dissociation (ETD) demonstrated successful identification of a total of 236 phosphorylation sites in the murine heart; 210 of these sites were novel. These 236 sites were mapped to 181 phosphoproteins and 203 phosphopeptides. Among those identified, 45 phosphorylation sites were captured only by CID, whereas 185 phosphorylation sites, including a novel modification on ubiquinol-cytochrome c reductase protein 1 (Ser-212), were identified only by ETD, underscoring the advantage of a combined CID and ETD approach. The biological significance of the cardiac mitochondrial phosphoproteome was evaluated. Our investigations illustrated key regulatory sites in murine cardiac mitochondrial pathways as targets of phosphorylation regulation, including components of the electron transport chain (ETC) complexes and enzymes involved in metabolic pathways (e.g. tricarboxylic acid cycle). Furthermore, calcium overload injured cardiac mitochondrial ETC function, whereas enhanced phosphorylation of ETC via application of phosphatase inhibitors restored calcium-attenuated ETC complex I and complex III activities, demonstrating positive regulation of ETC function by phosphorylation. Moreover, in silico analyses of the identified phosphopeptide motifs illuminated the molecular nature of participating kinases, which included several known mitochondrial kinases (e.g. pyruvate dehydrogenase kinase) as well as kinases whose mitochondrial location was not previously appreciated (e.g. Src). In conclusion, the phosphorylation events defined herein advance our understanding of cardiac mitochondrial biology, facilitating the integration of the still fragmentary knowledge about mitochondrial signaling networks, metabolic pathways, and intrinsic mechanisms of functional regulation in the heart.

Contrasting Proteome Biology and Functional Heterogeneity of the 20 S Proteasome Complexes in Mammalian Tissues

The 20 S proteasome complexes are major contributors to the intracellular protein degradation machinery in mammalian cells. Systematic administration of proteasome inhibitors to combat disease (e.g. cancer) has resulted in positive outcomes as well as adversary effects. The latter was attributed to, at least in part, a lack of understanding in the organ-specific responses to inhibitors and the potential diversity of proteomes of these complexes in different tissues. Accordingly, we conducted a proteomic study to characterize the 20 S proteasome complexes and their postulated organ-specific responses in the heart and liver. The cardiac and hepatic 20 S proteasomes were isolated from the same mouse strain with identical genetic background. We examined the molecular composition, complex assembly, post-translational modifications and associating partners of these proteasome complexes. Our results revealed an organ-specific molecular organization of the 20 S proteasomes with distinguished patterns of post-translational modifications as well as unique complex assembly characteristics. Furthermore, the proteome diversities are concomitant with a functional heterogeneity of the proteolytic patterns exhibited by these two organs. In particular, the heart and liver displayed distinct activity profiles to two proteasome inhibitors, epoxomicin and Z-Pro-Nle-Asp-H. Finally, the heart and liver demonstrated contrasting regulatory mechanisms from the associating partners of these proteasomes. The functional heterogeneity of the mammalian 20 S proteasome complexes underscores the concept of divergent proteomes among organs in the context of an identical genome.

Revealing the Dynamics of the 20 S Proteasome Phosphoproteome A Combined CID and Electron Transfer Dissociation Approach

The 20 S proteasomes play a critical role in intracellular homeostasis and stress response. Their function is tuned by covalent modifications, such as phosphorylation. In this study, we performed a comprehensive characterization of the phosphoproteome for the 20 S proteasome complexes in both the murine heart and liver. A platform combining parallel approaches in differential sample fractionation (SDS-PAGE, IEF, and two-dimensional electrophoresis), enzymatic digestion (trypsin and chymotrypsin), phosphopeptide enrichment (TiO2), and peptide fragmentation (CID and electron transfer dissociation (ETD)) has proven to be essential for identifying low abundance phosphopeptides. As a result, a total of 52 phosphorylation identifications were made in mammalian tissues; 44 of them were novel. These identifications include single (serine, threonine, and tyrosine) and dual phosphorylation peptides. 34 phosphopeptides were identified by CID; 10 phosphopeptides, including a key modification on the catalytically essential β5 subunit, were identified only by ETD; eight phosphopeptides were shared identifications by both CID and ETD. Besides the commonly shared phosphorylation sites, unique sites were detected in the murine heart and liver, documenting variances in phosphorylation between tissues within the proteasome populations. Furthermore the biological significance of these 20 S phosphoproteomes was evaluated. The role of cAMP-dependent protein kinase A (PKA) to modulate these phosphoproteomes was examined. Using a proteomics approach, many of the cardiac and hepatic 20 S subunits were found to be substrate targets of PKA. Incubation of the intact 20 S proteasome complexes with active PKA enhanced phosphorylation in both existing PKA phosphorylation sites as well as novel sites in these 20 S subunits. Furthermore treatment with active PKA significantly elevated all three peptidase activities (β1 caspase-like, β2 trypsin-like, and β5 chymotrypsin-like), demonstrating a functional role of PKA in governing these 20 S phosphoproteomes.

Altered Proteome Biology of Cardiac Mitochondria Under Stress Conditions

Myocardial ischemia-reperfusion induces mitochondrial dysfunction and, depending upon the degree of injury, may lead to cardiac cell death. However, our ability to understand mitochondrial dysfunction has been hindered by an absence of molecular markers defining the various degrees of injury. To address this paucity of knowledge, we sought to characterize the impact of ischemic damage on mitochondrial proteome biology. We hypothesized that ischemic injury induces differential alterations in various mitochondrial subcompartments, that these proteomic changes are specific to the severity of injury, and that they are important to subsequent cellular adaptations to myocardial ischemic injury. Accordingly, an in vitro model of cardiac mitochondria injury in mice was established to examine two stress conditions: reversible injury (induced by mild calcium overload) and irreversible injury (induced by hypotonic stimuli). Both forms of injury had a drastic impact on the proteome biology of cardiac mitochondria. Altered mitochondrial function was concomitant with significant protein loss/shedding from the injured organelles. In the setting of mild calcium overload, mitochondria retained functionality despite the release of numerous proteins, and the majority of mitochondria remained intact. In contrast, hypotonic stimuli caused severe damage to mitochondrial structure and function, induced increased oxidative modification of mitochondrial proteins, and brought about detrimental changes to the subproteomes of the inner mitochondrial membrane and matrix. Using an established in vivo murine model of regional myocardial ischemic injury, we validated key observations made by the in vitro model. This preclinical investigation provides function and suborganelle location information on a repertoire of cardiac mitochondrial proteins sensitive to ischemia reperfusion stress and highlights protein clusters potentially involved in mitochondrial dysfunction in the setting of ischemic injury.

Two-dimensional electrophoresis-based characterization of post-translational modifications of mammalian 20S proteasome complexes

PTMs serve as key regulatory mechanisms for 20S proteasome functions. Alterations in 20S PTMs have been previously observed with changes in modified protein degradation patterns and altered cellular phenotypes. Despite decades of investigation, our knowledge pertaining to the various PTMs of 20S complexes and their biological significance remain limited. In this investigation, we show that 2‐DE offers an analytical tool with high resolution and reproducibility. Accordingly, it has been applied for the characterization of PTMs including glycosylation, phosphorylation, oxidation, and nitrosylation. The PTMs of murine cardiac 20S proteasomes and their associating proteins were examined. Our 2‐DE analyses displayed over 25 spots for the 20S complexes (17 subunits), indicating multiply modified subunits of cardiac proteasomes. The identification of specific PTM sites subsequent to 2‐DE was supported by MS. These PTMs included phosphorylation and oxidation. Most of the PTMs occurred in low stoichiometry and required enrichment to enhance the detection sensitivity. In conclusion, our studies support 2‐DE as a central tool in the analyses of 20S proteasome PTMs. The approaches utilized in this investigation demonstrate their application in mapping the PTMs of the 20S proteasomes in cardiac tissue, which are applicable to other samples and biological conditions.

Sheathless capillary electrophoresis-tandem mass spectrometry for top-down characterization of Pyrococcus furiosus proteins on a proteome scale.

Intact protein analysis via top-down mass spectrometry (MS) provides the unique capability of fully characterizing protein isoforms and combinatorial post-translational modifications (PTMs) compared to the bottom-up MS approach. Front-end protein separation poses a challenge for analyzing complex mixtures of intact proteins on a proteomic scale. Here we applied capillary electrophoresis (CE) through a sheathless capillary electrophoresis-electrospray ionization (CESI) interface coupled to an Orbitrap Elite mass spectrometer to profile the proteome from Pyrococcus furiosus. CESI-top-down MS analysis of Pyrococcus furiosus cell lysate identified 134 proteins and 291 proteoforms with a total sample consumption of 270 ng in 120 min of total analysis time. Truncations and various PTMs were detected, including acetylation, disulfide bonds, oxidation, glycosylation, and hypusine. This is the largest scale analysis of intact proteins by CE-top-down MS to date.

Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane

Endocytic protein trafficking is directed by sorting signals on cargo molecules that are recognized by cytosolic adaptor proteins. However, the steps necessary to segregate the variety of cargoes during endocytosis remain poorly defined. Using Caenorhabditis elegans, we demonstrate that multiple plasma membrane endocytic adaptors function redundantly to regulate clathrin-mediated endocytosis and to recruit components of the endosomal sorting complex required for transport (ESCRT) machinery to the cell surface to direct the sorting of ubiquitin-modified substrates. Moreover, our data suggest that preassembly of cargoes with the ESCRT-0 complex at the plasma membrane enhances the efficiency of downstream sorting events in the endolysosomal system. In the absence of a heterooligomeric adaptor complex composed of FCHO, Eps15, and intersectin, ESCRT-0 accumulation at the cell surface is diminished, and the degradation of a ubiquitin-modified cargo slows significantly without affecting the rate of its clathrin-mediated internalization. Consistent with a role for the ESCRT machinery during cargo endocytosis, we further show that the ESCRT-0 complex accumulates at a subset of clathrin-coated pits on the surface of human cells. Our findings suggest a unique mechanism by which ubiquitin-modified cargoes are sequestered into the endolysosomal pathway.

Nudel/NudE and Lis1 promote dynein and dynactin interaction in the context of spindle morphogenesis

Nudel/NudE facilitates the binding of Lis1 to dynein, which subsequently enhances the recruitment of dynactin to dynein, and dynactin antagonizes Lis1 to relieve Lis1-induced dynein stall on microtubules.