Proteomics of the heart.

Mass spectrometry-based proteomics is a sophisticated identification tool specializing in portraying protein dynamics at a molecular level. Proteomics provides biologists with a snapshot of context-dependent protein and proteoform expression, structural conformations, dynamic turnover, and protein-protein interactions. Cardiac proteomics can offer a broader and deeper understanding of the molecular mechanisms that underscore cardiovascular disease, and it is foundational to the development of future therapeutic interventions. This review encapsulates the evolution, current technologies, and future perspectives of proteomic-based mass spectrometry as it applies to the study of the heart. Key technological advancements have allowed researchers to study proteomes at a single-cell level and employ robot-assisted automation systems for enhanced sample preparation techniques, and the increase in fidelity of the mass spectrometers has allowed for the unambiguous identification of numerous dynamic posttranslational modifications. Animal models of cardiovascular disease, ranging from early animal experiments to current sophisticated models of heart failure with preserved ejection fraction, have provided the tools to study a challenging organ in the laboratory. Further technological development will pave the way for the implementation of proteomics even closer within the clinical setting, allowing not only scientists but also patients to benefit from an understanding of protein interplay as it relates to cardiac disease physiology.

Automated proteomic sample preparation: The key component for high throughput and quantitative mass spectrometry analysis.

Sample preparation for mass spectrometry-based proteomics has many tedious and time-consuming steps that can introduce analytical errors. In particular, the steps around the proteolytic digestion of protein samples are prone to inconsistency. One route for reliable sample processing is the development and optimization of a workflow utilizing an automated liquid handling workstation. Diligent assessment of the sample type, protocol design, reagents, and incubation conditions can significantly improve the speed and consistency of preparation. When combining robust liquid chromatography-mass spectrometry with either discovery or targeted methods, automated sample preparation facilitates increased throughput and reproducible quantitation of biomarker candidates. These improvements in analysis are also essential to process the large patient cohorts necessary to validate a candidate biomarker for potential clinical use. This article reviews the steps in the workflow, optimization strategies, and known applications in clinical, pharmaceutical, and research fields that demonstrate the broad utility for improved automation of sample preparation in the proteomic field.

S-Nitrosoglutathione Reductase Deficiency Causes Aberrant Placental S-Nitrosylation and Preeclampsia.

Background Preeclampsia, a leading cause of maternal and fetal mortality and morbidity, is characterized by an increase in S-nitrosylated proteins and reactive oxygen species, suggesting a pathophysiologic role for dysregulation in nitrosylation and nitrosative stress. Methods and Results Here, we show that mice lacking S-nitrosoglutathione reductase (GSNOR-/-), a denitrosylase regulating protein S-nitrosylation, exhibit a preeclampsia phenotype, including hypertension, proteinuria, renal pathology, cardiac concentric hypertrophy, decreased placental vascularization, and fetal growth retardation. Reactive oxygen species, NO, and peroxynitrite levels are elevated. Importantly, mass spectrometry reveals elevated placental S-nitrosylated amino acid residues in GSNOR-/- mice. Ascorbate reverses the phenotype except for fetal weight, reduces the difference in the S-nitrosoproteome, and identifies a unique set of S-nitrosylated proteins in GSNOR-/- mice. Importantly, human preeclamptic placentas exhibit decreased GSNOR activity and increased nitrosative stress. Conclusions Therefore, deficiency of GSNOR creates dysregulation of placental S-nitrosylation and preeclampsia in mice, which can be rescued by ascorbate. Coupled with similar findings in human placentas, these findings offer valuable insights and therapeutic implications for preeclampsia.

Standardized Workflow for Precise Mid- and High-Throughput Proteomics of Blood Biofluids.

BACKGROUND: Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that can accommodate 3 blood-based proteomes: naive plasma, depleted plasma and dried blood. METHODS: Optimal conditions for sample preparation and data independent acquisition-mass spectrometry analysis were established in plasma then automated for depleted plasma and dried blood. The mass spectrometry workflow was modified to facilitate sensitive high-throughput analysis or deeper profiling with mid-throughput analysis. Analytical performance was evaluated by the linear response of peptides and proteins to a 6- or 7-point dilution curve and the reproducibility of the relative peptide and protein intensity for 5 digestion replicates per day on 3 different days for each biofluid. RESULTS: Using the high-throughput workflow, 74% (plasma), 93% (depleted), and 87% (dried blood) displayed an inter-day CV <30%. The mid-throughput workflow had 67% (plasma), 90% (depleted), and 78% (dried blood) of peptides display an inter-day CV <30%. Lower limits of detection and quantification were determined for peptides and proteins observed in each biofluid and workflow. Based on each protein and peptide's analytical performance, we could describe the observable, reliable, reproducible, and quantifiable proteomes for each biofluid and workflow. CONCLUSION: The standardized workflows established here allows for reproducible and quantifiable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow should simplify discovery approaches and facilitate the translation of candidate markers into clinical use.

Multi-modal normalization of resting-state using local physiology reduces changes in functional connectivity patterns observed in mTBI patients.

Blood oxygenation level dependent (BOLD) resting-state functional magnetic resonance imaging (rs-fMRI) may serve as a sensitive marker to identify possible changes in the architecture of large-scale networks following mild traumatic brain injury (mTBI). Differences in functional connectivity (FC) measurements derived from BOLD rs-fMRI may however be confounded by changes in local cerebrovascular physiology and neurovascular coupling mechanisms, without changes in the underlying neuronally driven connectivity of networks. In this study, multi-modal neuroimaging data including BOLD rs-fMRI, baseline cerebral blood flow (CBF0) and cerebrovascular reactivity (CVR; acquired using a hypercapnic gas breathing challenge) were collected in 23 subjects with reported mTBI (14.6±14.9 months post-injury) and 27 age-matched healthy controls. Despite no group differences in CVR within the networks of interest (P > 0.05, corrected), significantly higher CBF0 was documented in the mTBI subjects (P < 0.05, corrected), relative to the controls. A normalization method designed to account for differences in CBF0 post-mTBI was introduced to evaluate the effects of such an approach on reported group differences in network connectivity. Inclusion of regional perfusion measurements in the computation of correlation coefficients within and across large-scale networks narrowed the differences in FC between the groups, suggesting that this approach may elucidate unique changes in connectivity post-mTBI while accounting for shared variance with CBF0. Altogether, our results provide a strong paradigm supporting the need to account for changes in physiological modulators of BOLD in order to expand our understanding of the effects of brain injury on large-scale FC of cortical networks.

IKs ion-channel pore conductance can result from individual voltage sensor movements.

The IKs current has an established role in cardiac action potential repolarization, and provides a repolarization reserve at times of stress. The underlying channels are formed from tetramers of KCNQ1 along with one to four KCNE1 accessory subunits, but how these components together gate the IKs complex to open the pore is controversial. Currently, either a concerted movement involving all four subunits of the tetramer or allosteric regulation of open probability through voltage-dependent subunit activation is thought to precede opening. Here, by using the E160R mutation in KCNQ1 or the F57W mutation in KCNE1 to prevent or impede, respectively, voltage sensors from moving into activated conformations, we demonstrate that a concerted transition of all four subunits after voltage sensor activation is not required for the opening of IKs channels. Tracking voltage sensor movement, via [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) modification and fluorescence recordings, shows that E160R-containing voltage sensors do not translocate upon depolarization. E160R, when expressed in all four KCNQ1 subunits, is nonconducting, but if one, two, or three voltage sensors contain the E160R mutation, whole-cell and single-channel currents are still observed in both the presence and absence of KCNE1, and average conductance is reduced proportional to the number of E160R voltage sensors. The data suggest that KCNQ1 + KCNE1 channels gate like KCNQ1 alone. A model of independent voltage sensors directly coupled to open states can simulate experimental changes in IKs current kinetics, including the nonlinear depolarization of the conductance-voltage (G-V) relationship, and tail current acceleration as the number of nonactivatable E160R subunits is increased.

Precision Profiling of the Cardiovascular Post-Translationally Modified Proteome: Where There Is a Will, There Is a Way.

There is an exponential increase in biological complexity as initial gene transcripts are spliced, translated into amino acid sequence, and post-translationally modified. Each protein can exist as multiple chemical or sequence-specific proteoforms, and each has the potential to be a critical mediator of a physiological or pathophysiological signaling cascade. Here, we provide an overview of how different proteoforms come about in biological systems and how they are most commonly measured using mass spectrometry-based proteomics and bioinformatics. Our goal is to present this information at a level accessible to every scientist interested in mass spectrometry and its application to proteome profiling. We will specifically discuss recent data linking various protein post-translational modifications to cardiovascular disease and conclude with a discussion for enablement and democratization of proteomics across the cardiovascular and scientific community. The aim is to inform and inspire the readership to explore a larger breadth of proteoform, particularity post-translational modifications, related to their particular areas of expertise in cardiovascular physiology.

A Proteomics Workflow for Dual Labeling Biotin Switch Assay to Detect and Quantify Protein S-Nitroylation.

S-nitrosylation (or S-nitrosation, SNO) is an oxidative posttranslational modification to the thiol group of a cysteine amino acid residue. There are several methods to detect SNO modifications, mostly based on the classic biotin-switch assay, where the labile SNO sites are replaced with a stable biotin moiety to facilitate enrichment of the modified proteins. As the technique has evolved, new and more advanced thiol-reactive reagents have been introduced in the protocol to improve the identification of modified peptides or to quantify the level of modification at individual cysteine residues. However, the growing diversity of thiol-reactive affinity tags has not produced a consistent set of protein modifications, suggesting incomplete coverage using a single tag. Here, we present a parallel dual labeling strategy followed by an optimized proteomics workflow, which maximizes the overall detection of SNO by reducing the labeling bias derived from the use of a single tag-capture approach.

Photo-Cross-Linking of IKs Demonstrates State-Dependent Interactions between KCNE1 and KCNQ1.

The slow delayed rectifier potassium current (IKs) is a key repolarizing current during the cardiac action potential. It consists of four KCNQ1 α-subunits and up to four KCNE1 ß-subunits, which are thought to reside within external clefts of the channel. The interaction of KCNE1 with KCNQ1 dramatically delays opening of the channel but the mechanisms by which this occur are not yet fully understood. Here, we have used unnatural amino acid photo-cross-linking to investigate the dynamic interactions that occur between KCNQ1 and KCNE1 during activation gating. The unnatural amino acid p-Benzoylphenylalanine was successfully incorporated into two residues within the transmembrane domain of KCNE1: F56 and F57. UV-induced cross-linking suggested that F56Bpa interacts with KCNQ1 in the open state, whereas F57Bpa interacts predominantly in resting channel conformations. When UV was applied at progressively more depolarized preopen holding potentials, cross-linking of F57Bpa with KCNQ1 was slowed, which indicates that KCNE1 is displaced within the channel's cleft early during activation, or that conformational changes in KCNQ1 alter its interaction with KCNE1. In E1R/R4E KCNQ1, a mutant with constitutively activated voltage sensors, F56Bpa and F57Bpa KCNE1 were cross-linked in open and closed states, respectively, which suggests that their actions are mediated mainly by modulation of KCNQ1 pore function.

Unnatural amino acid photo-crosslinking of the IKs channel complex demonstrates a KCNE1:KCNQ1 stoichiometry of up to 4:4.

Cardiac repolarization is determined in part by the slow delayed rectifier current (IKs), through the tetrameric voltage-gated ion channel, KCNQ1, and its ß-subunit, KCNE1. The stoichiometry between α and ß-subunits has been controversial with studies reporting either a strict 2 KCNE1:4 KCNQ1 or a variable ratio up to 4:4. We used IKs fusion proteins linking KCNE1 to one (EQ), two (EQQ) or four (EQQQQ) KCNQ1 subunits, to reproduce compulsory 4:4, 2:4 or 1:4 stoichiometries. Whole cell and single-channel recordings showed EQQ and EQQQQ to have increasingly hyperpolarized activation, reduced conductance, and shorter first latency of opening compared to EQ - all abolished by the addition of KCNE1. As well, using a UV-crosslinking unnatural amino acid in KCNE1, we found EQQQQ and EQQ crosslinking rates to be progressively slowed compared to KCNQ1, which demonstrates that no intrinsic mechanism limits the association of up to four ß-subunits within the IKs complex.

Dual Labeling Biotin Switch Assay to Reduce Bias Derived From Different Cysteine Subpopulations: A Method to Maximize S-Nitrosylation Detection.

RATIONALE: S-nitrosylation (SNO), an oxidative post-translational modification of cysteine residues, responds to changes in the cardiac redox-environment. Classic biotin-switch assay and its derivatives are the most common methods used for detecting SNO. In this approach, the labile SNO group is selectively replaced with a single stable tag. To date, a variety of thiol-reactive tags have been introduced. However, these methods have not produced a consistent data set, which suggests an incomplete capture by a single tag and potentially the presence of different cysteine subpopulations. OBJECTIVE: To investigate potential labeling bias in the existing methods with a single tag to detect SNO, explore if there are distinct cysteine subpopulations, and then, develop a strategy to maximize the coverage of SNO proteome. METHODS AND RESULTS: We obtained SNO-modified cysteine data sets for wild-type and S-nitrosoglutathione reductase knockout mouse hearts (S-nitrosoglutathione reductase is a negative regulator of S-nitrosoglutathione production) and nitric oxide-induced human embryonic kidney cell using 2 labeling reagents: the cysteine-reactive pyridyldithiol and iodoacetyl based tandem mass tags. Comparison revealed that <30% of the SNO-modified residues were detected by both tags, whereas the remaining SNO sites were only labeled by 1 reagent. Characterization of the 2 distinct subpopulations of SNO residues indicated that pyridyldithiol reagent preferentially labels cysteine residues that are more basic and hydrophobic. On the basis of this observation, we proposed a parallel dual-labeling strategy followed by an optimized proteomics workflow. This enabled the profiling of 493 SNO sites in S-nitrosoglutathione reductase knockout hearts. CONCLUSIONS: Using a protocol comprising 2 tags for dual-labeling maximizes overall detection of SNO by reducing the previously unrecognized labeling bias derived from different cysteine subpopulations.

Quantification of mitochondrial S-nitrosylation by CysTMT6 switch assay.

S-nitrosylation (SNO) is an important oxidative posttranslational modification in the regulation of cardiac mitochondria. SNO modification of several mitochondrial proteins has been associated with cardiac preconditioning and improved cell survival following ischemia/reperfusion injury. Due to their labile nature, SNO modifications are challenging to study using traditional biochemical techniques; particularly, the identification of individual modified cysteine residues. Here, we describe the details of the cysTMT(6) switch assay, a variation of the classic biotin switch protocol. The cysTMT(6) reagent provides a simplified and powerful approach to SNO detection by combining unambiguous identification of the modified cysteine residue and relative quantification of up to six samples by mass spectrometry analysis.

Redox regulation of mitochondrial ATP synthase.

Reversible cysteine oxidative post-translational modifications (Ox-PTMs) represent an important mechanism to regulate protein structure and function. In mitochondria, redox reactions can modulate components of the electron transport chain (ETC), the F(1)F(0)-ATP synthase complex, and other matrix proteins/enzymes. Emerging evidence has linked Ox-PTMs to mitochondrial dysfunction and heart failure, highlighting some potential therapeutic avenues. Ox-PTMs can modify a variety of amino acid residues, including cysteine, and have the potential to modulate the function of a large number of proteins. Among this group, there is a selected subset of amino acid residues that can function as redox switches. These unique sites are proposed to monitor the cell's oxidative balance through their response to the various Ox-PTMs. In this review, the role of Ox-PTMs in the regulation of the F(1)F(0)-ATP synthase complex is discussed in the context of heart failure and its possible clinical treatment.

Cysteine oxidative posttranslational modifications: emerging regulation in the cardiovascular system.

In the cardiovascular system, changes in oxidative balance can affect many aspects of cellular physiology through redox-signaling. Depending on the magnitude, fluctuations in the cell's production of reactive oxygen and nitrogen species can regulate normal metabolic processes, activate protective mechanisms, or be cytotoxic. Reactive oxygen and nitrogen species can have many effects including the posttranslational modification of proteins at critical cysteine thiols. A subset can act as redox-switches, which elicit functional effects in response to changes in oxidative state. Although the general concepts of redox-signaling have been established, the identity and function of many regulatory switches remains unclear. Characterizing the effects of individual modifications is the key to understand how the cell interprets oxidative signals under physiological and pathological conditions. Here, we review the various cysteine oxidative posttranslational modifications and their ability to function as redox-switches that regulate the cell's response to oxidative stimuli. In addition, we discuss how these modifications have the potential to influence other posttranslational modifications' signaling pathways though cross-talk. Finally, we review the increasing number of tools being developed to identify and quantify the various cysteine oxidative posttranslational modifications and how this will advance our understanding of redox-regulation.

Nitroxyl-mediated disulfide bond formation between cardiac myofilament cysteines enhances contractile function.

RATIONALE: In the myocardium, redox/cysteine modification of proteins regulating Ca(2+) cycling can affect contraction and may have therapeutic value. Nitroxyl (HNO), the one-electron-reduced form of nitric oxide, enhances cardiac function in a manner that suggests reversible cysteine modifications of the contractile machinery. OBJECTIVE: To determine the effects of HNO modification in cardiac myofilament proteins. METHODS AND RESULTS: The HNO-donor, 1-nitrosocyclohexyl acetate, was found to act directly on the myofilament proteins, increasing maximum force (F(max)) and reducing the concentration of Ca(2+) for 50% activation (Ca(50)) in intact and skinned cardiac muscles. The effects of 1-nitrosocyclohexyl acetate are reversible by reducing agents and distinct from those of another HNO donor, Angeli salt, which was previously reported to increase F(max) without affecting Ca50. Using a new mass spectrometry capture technique based on the biotin switch assay, we identified and characterized the formation by HNO of a disulfide-linked actin-tropomyosin and myosin heavy chain-myosin light chain 1. Comparison of the 1-nitrosocyclohexyl acetate and Angeli salt effects with the modifications induced by each donor indicated the actin-tropomyosin and myosin heavy chain-myosin light chain 1 interactions independently correlated with increased Ca(2+) sensitivity and force generation, respectively. CONCLUSIONS: HNO exerts a direct effect on cardiac myofilament proteins increasing myofilament Ca(2+) responsiveness by promoting disulfide bond formation between critical cysteine residues. These findings indicate a novel, redox-based modulation of the contractile apparatus, which positively impacts myocardial function, providing further mechanistic insight for HNO as a therapeutic agent.

Identification of novel interaction between annexin A2 and keratin 17: evidence for reciprocal regulation.

Keratins are cytoplasmic intermediate filament proteins providing crucial structural support in epithelial cells. Keratin expression has diagnostic and even prognostic value in disease settings, and recent studies have uncovered modulatory roles for select keratin proteins in signaling pathways regulating cell growth and cell death. Elevated keratin expression in select cancers is correlated with higher expression of EGF receptor (EGFR), whose overexpression and/or mutation give rise to cancer. To explore the role of keratins in oncogenic signaling pathways, we examined the regulation of epithelial growth-associated keratin 17 (K17) in response to EGFR activation. K17 is specifically up-regulated in detergent-soluble fraction upon EGFR activation, and immunofluorescence analysis revealed alterations in K17-containing filaments. Interestingly, we identified AnxA2 as a novel interacting partner of K17, and this interaction is antagonized by EGFR activation. K17 and AnxA2 proteins show reciprocal regulation. Modulating expression of AnxA2 altered K17 stability, and AnxA2 overexpression delays EGFR-mediated change in K17 detergent solubility. Down-regulation of K17 expression, in turn, results in decreased AnxA2 phosphorylation at Tyr-23. These findings uncover a novel interaction involving K17 and AnxA2 and identify AnxA2 as a potential regulator of keratin filaments.

Identification and quantification of S-nitrosylation by cysteine reactive tandem mass tag switch assay.

Redox-switches are critical cysteine thiols that are modified in response to changes in the cell's environment conferring a functional effect. S-nitrosylation (SNO) is emerging as an important modulator of these regulatory switches; however, much remains unknown about the nature of these specific cysteine residues and how oxidative signals are interpreted. Because of their labile nature, SNO-modifications are routinely detected using the biotin switch assay. Here, a new isotope coded cysteine thiol-reactive multiplex reagent, cysTMT(6), is used in place of biotin, for the specific detection of SNO-modifications and determination of individual protein thiol-reactivity. S-nitrosylation was measured in human pulmonary arterial endothelia cells in vitro and in vivo using the cysTMT(6) quantitative switch assay coupled with mass spectrometry. Cell lysates were treated with S-nitrosoglutathione and used to identify 220 SNO-modified cysteines on 179 proteins. Using this approach it was possible to discriminate potential artifacts including instances of reduced protein disulfide bonds (6) and S-glutathionylation (5) as well as diminished ambiguity in site assignment. Quantitative analysis over a range of NO-donor concentrations (2, 10, 20 µm; GSNO) revealed a continuum of reactivity to SNO-modification. Cysteine response was validated in living cells, demonstrating a greater number of less sensitive cysteine residues are modified with increasing oxidative stimuli. Of note, the majority of available cysteines were found to be unmodified in the current treatment suggesting significant additional capacity for oxidative modifications. These results indicate a possible mechanism for the cell to gauge the magnitude of oxidative stimuli through the progressive and specific accumulation of modified redox-switches.

Phosphodiesterase-5A (PDE5A) is localized to the endothelial caveolae and modulates NOS3 activity.

AIMS: It has been well demonstrated that phosphodiesterase-5A (PDE5A) is expressed in smooth muscle cells and plays an important role in regulation of vascular tone. The role of endothelial PDE5A, however, has not been yet characterized. The present study was undertaken to determine the presence, localization, and potential physiologic significance of PDE5A within vascular endothelial cells. METHODS AND RESULTS: We demonstrate primary location of human, mouse, and bovine endothelial PDE5A at or near caveolae. We found that the spatial localization of PDE5A at the level of caveolin-rich lipid rafts allows for a feedback loop between endothelial PDE5A and nitric oxide synthase (NOS3). Treatment of human endothelium with PDE5A inhibitors resulted in a significant increase in NOS3 activity, whereas overexpression of PDE5A using an adenoviral vector, both in vivo and in cell culture, resulted in decreased NOS3 activity and endothelium-dependent vasodilation. The molecular mechanism responsible for these interactions is primarily regulated by cGMP-dependent second messenger. PDE5A overexpression also resulted in a significant decrease in protein kinase 1 (PKG1) activity. Overexpression of PKG1 rapidly activated NOS3, whereas silencing of the PKG1 gene with siRNA inhibited both NOS3 phosphorylation (S1179) and activity, indicating a novel role for PKG1 in direct regulation of NOS3. CONCLUSION: Our data collectively suggest another target for PDE5A inhibition in endothelial dysfunction and provide another physiologic significance for PDE5A in the modulation of endothelial-dependent flow-mediated vasodilation. Using both in vitro and in vivo models, as well as human data, we show that inhibition of endothelial PDE5A improves endothelial function.