Determination of promising inhibitors for N-SH2 domain of SHP2 tyrosine phosphatase: an in silico study.

There are many genes that produce proteins related to diseases and these proteins can be targeted with drugs as a potential therapeutic approach. Recent advancement in drug discovery techniques have created new opportunities for treating variety of diseases by targeting disease-related proteins. Structure-based drug discovery is a faster and more cost-effective approach than traditional methods. SHP2 phosphatase, encoded by the PTPN11 gene, has been the focus of much attention due to its involvement in many types of diseases. The biological function of SHP2 is enabled mostly by protein-protein interaction through its SH2 domains. In this study, we report the identification of a potential small molecule inhibitor for the N-SH2 domain of SHP2 by structure-based drug discovery approach. We utilized molecular docking studies, followed by molecular dynamics simulations and MM/PBSA calculations, to analyze compounds retrieved from the Broad's Drug Repurposing Hub and ZINC15 databases. We selected 10 hit compounds with the best docking scores from the libraries and examined their binding properties in the N-SH2 domain. We found that compound CID 60838 (Irinotecan) was the most suitable compound with a binding free energy value of - 64.45 kcal/mol and significant interactions with the target residues in the domain.

Dual Targeting of DNA Damage Response Proteins Implicated in Cancer Radioresistance.

Ionizing radiation can induce different types of DNA lesions, leading to genomic instability and ultimately cell death. Radiation therapy or radiotherapy, a major modality in cancer treatment, harnesses the genotoxic potential of radiation to target and destroy cancer cells. Nevertheless, cancer cells have the capacity to develop resistance to radiation treatment (radioresistance), which poses a major obstacle in the effective management of cancer. It has been shown that administration of platinum-based drugs to cancer patients can increase tumor radiosensitivity, but despite this, it is associated with severe adverse effects. Several lines of evidence support that activation of the DNA damage response and repair machinery in the irradiated cancer cells enhances radioresistance and cellular survival through the efficient repair of DNA lesions. Therefore, targeting of key DNA damage repair factors would render cancer cells vulnerable to the irradiation effects, increase cancer cell killing, and reduce the risk of side effects on healthy tissue. Herein, we have employed a computer-aided drug design approach for generating ab initio a chemical compound with drug-like properties potentially targeting two proteins implicated in multiple DNA repair pathways. The findings of this study could be taken into consideration in clinical decision-making in terms of co-administering radiation with DNA damage repair factor-based drugs.

TRAIL promotes the polarization of human macrophages toward a proinflammatory M1 phenotype and is associated with increased survival in cancer patients with high tumor macrophage content.

Background: TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can either induce cell death or activate survival pathways after binding to death receptors (DRs) DR4 or DR5. TRAIL is investigated as a therapeutic agent in clinical trials due to its selective toxicity to transformed cells. Macrophages can be polarized into pro-inflammatory/tumor-fighting M1 macrophages or anti-inflammatory/tumor-supportive M2 macrophages and an imbalance between M1 and M2 macrophages can promote diseases. Therefore, identifying modulators that regulate macrophage polarization is important to design effective macrophage-targeted immunotherapies. The impact of TRAIL on macrophage polarization is not known. Methods: Primary human monocyte-derived macrophages were pre-treated with either TRAIL or with DR4 or DR5-specific ligands and then polarized into M1, M2a, or M2c phenotypes in vitro. The expression of M1 and M2 markers in macrophage subtypes was analyzed by RNA sequencing, qPCR, ELISA, and flow cytometry. Furthermore, the cytotoxicity of the macrophages against U937 AML tumor targets was assessed by flow cytometry. TCGA datasets were also analyzed to correlate TRAIL with M1/M2 markers, and the overall survival of cancer patients. Results: TRAIL increased the expression of M1 markers at both mRNA and protein levels while decreasing the expression of M2 markers at the mRNA level in human macrophages. TRAIL also shifted M2 macrophages towards an M1 phenotype. Our data showed that both DR4 and DR5 death receptors play a role in macrophage polarization. Furthermore, TRAIL enhanced the cytotoxicity of macrophages against the AML cancer cells in vitro. Finally, TRAIL expression was positively correlated with increased expression of M1 markers in the tumors from ovarian and sarcoma cancer patients and longer overall survival in cases with high, but not low, tumor macrophage content. Conclusions: TRAIL promotes the polarization of human macrophages toward a proinflammatory M1 phenotype via both DR4 and DR5. Our study defines TRAIL as a new regulator of macrophage polarization and suggests that targeting DRs can enhance the anti-tumorigenic response of macrophages in the tumor microenvironment by increasing M1 polarization.

Synthesis and Comprehensive in Vivo Activity Profiling of Olean-12-en-28-ol, 3ß-Pentacosanoate in Experimental Autoimmune Encephalomyelitis: A Natural Remyelinating and Anti-Inflammatory Agent.

Multiple sclerosis (MS) treatment has received much attention, yet there is still no certain cure. We herein investigate the therapeutic effect of olean-12-en-28-ol, 3ß-pentacosanoate (OPCA) on a preclinical model of MS. First, OPCA was synthesized semisynthetically and characterized. Then, the mice with MOG35-55-induced experimental autoimmune/allergic encephalomyelitis (EAE) were given OPCA along with a reference drug (FTY720). Biochemical, cellular, and molecular analyses were performed in serum and brain tissues to measure anti-inflammatory and neuroprotective responses. OPCA treatment protected EAE-induced changes in mouse brains maintaining blood-brain barrier integrity and preventing inflammation. Moreover, the protein and mRNA levels of MS-related genes such as HLD-DR1, CCL5, TNF-α, IL6, and TGFB1 were significantly reduced in OPCA-treated mouse brains. Notably, the expression of genes, including PLP, MBP, and MAG, involved in the development and structure of myelin was significantly elevated in OPCA-treated EAE. Furthermore, therapeutic OPCA effects included a substantial reduction in pro-inflammatory cytokines in the serum of treated EAE animals. Lastly, following OPCA treatment, the promoter regions for most inflammatory regulators were hypermethylated. These data support that OPCA is a valuable and appealing candidate for human MS treatment since OPCA not only normalizes the pro- and anti-inflammatory immunological bias but also stimulates remyelination in EAE.

Biochemical, pharmacological, and toxicological attributes of caper (Capparis ovata) flowering buds and berries pickles.

Capparis ovata is a natural plant that grows widely in Turkey and its flowering buds and berry pickle are used in traditional medicine. Thus, the current study was expanded to evaluate the biochemical, pharmacological, and toxicological aspects of the Capparis ovata water extract (COWE). To determine the biochemical properties of COWE, mineral and fatty acid content, elemental analysis, flavonoid/phenolic content, radical-scavenging capacity, and pesticide analysis were performed. Furthermore, to find out whether it had anti-inflammatory properties, reverse transcription-polymerase chain reaction (RT-PCR) and nuclear factor kappa B (NF-κB) luciferase activity tests were conducted. Whole-genome transcriptomic profiling was carried out at a dose level of 500 mg/kg COWE to understand its pharmacological effect. Transaminases in serum were tested, and quantitative polymerase chain reaction (qPCR) was done using a custom design array that included the stress and molecular toxicology pathway to establish its toxicological qualities. As a result of the evaluations, it was observed that COWE has a high mineral and unsaturated fatty acid content, flavonoid/phenolic content, and radical-scavenging ability. It significantly inhibited NF-κB transcriptional activity as well as inflammatory cytokine expression in T-lymphoblast cells. Whole-genome transcriptomic profiling depicted that COWE modulates immune responses by upregulating natural killer cell activation, cellular response to type I interferon, B-cell proliferation and differentiation, and Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways. Molecular Toxicology Pathfinder RT2 Profiler PCR array analysis revealed that COWE at or lower dose of 500 mg/kg/day did not cause a comparatively adverse effect. According to the findings, COWE is a rich source of nutrients and can be used as an adjunct therapy for various inflammatory diseases.

A subtractive proteomics approach for the identification of immunodominant Acinetobacter baumannii vaccine candidate proteins.

Background: Acinetobacter baumannii is one of the most life-threatening multidrug-resistant pathogens worldwide. Currently, 50%-70% of clinical isolates of A. baumannii are extensively drug-resistant, and available antibiotic options against A. baumannii infections are limited. There is still a need to discover specific de facto bacterial antigenic proteins that could be effective vaccine candidates in human infection. With the growth of research in recent years, several candidate molecules have been identified for vaccine development. So far, no public health authorities have approved vaccines against A. baumannii. Methods: This study aimed to identify immunodominant vaccine candidate proteins that can be immunoprecipitated specifically with patients' IgGs, relying on the hypothesis that the infected person's IgGs can capture immunodominant bacterial proteins. Herein, the outer-membrane and secreted proteins of sensitive and drug-resistant A. baumannii were captured using IgGs obtained from patient and healthy control sera and identified by Liquid Chromatography- Tandem Mass Spectrometry (LC-MS/MS) analysis. Results: Using the subtractive proteomic approach, we determined 34 unique proteins captured only in drug-resistant A. baumannii strain via patient sera. After extensively evaluating the predicted epitope regions, solubility, transverse membrane characteristics, and structural properties, we selected several notable vaccine candidates. Conclusion: We identified vaccine candidate proteins that triggered a de facto response of the human immune system against the antibiotic-resistant A. baumannii. Precipitation of bacterial proteins via patient immunoglobulins was a novel approach to identifying the proteins that could trigger a response in the patient immune system.

Protocol for cell surface biotinylation of magnetic labeled and captured human peripheral blood mononuclear cells.

Analysis of the surfaceome of a blood cell subset requires cell sorting, followed by surface protein enrichment. Here, we present a protocol combining magnetically activated cell sorting (MACS) and surface biotinylation of the target cell subset from human peripheral blood mononuclear cells (PBMCs). We describe the steps for isolating target cells and their in-column surface biotinylation, followed by isolation and mass spectrometry analysis of biotinylated proteins. The protocol enables in-column surface biotinylation of specific cell subsets with minimal membrane disruption.

Proteomic fertility markers in ram sperm.

Precise estimation of ram fertility is important for sheep farming to sustain reproduction efficiency and profitability of production. There, however, is no conventional method to accurately predict ram fertility. The objective of this study, therefore, was to ascertain proteomic profiles of ram sperm having contrasting fertility phenotypes. Mature rams (n = 66) having greater pregnancy rates than average (89.4 ± 7.2%) were assigned into relatively-greater fertility (GF; n = 31; 94.5 ± 2.8%) whereas those with less-than-average pregnancy rates were assigned into a lesser-fertility (LF; n = 25; 83.1 ± 5.73%; P = 0.028) group. Sperm samples from the outlier greatest- and least-fertility rams (n = 6, pregnancy rate; 98.4 ± 1.8% and 76.1 ± 3.9%) were used for proteomics assessments utilizing Label-free LC-MS/MS. A total of 997 proteins were identified, and among these, 840 were shared by both groups, and 57 and 93 were unique to GF and LF, respectively. Furthermore, 190 differentially abundant proteins were identified; the abundance of 124 was larger in GF while 66 was larger in LF rams. The GF ram sperm had 79 GO/pathway terms in ten major biological networks while there were 47 GO/pathway terms in six biological networks in sperm of LF rams. Accordingly, differential abundances of sperm proteins between sperm of GF and LF rams were indicative of functional implications of sperm proteome on male fertility. The results of this study emphasize there are potential protein markers for evaluation of semen quality and estimation of ram sperm fertilizing capacity.

Why Do Muse Stem Cells Present an Enduring Stress Capacity? Hints from a Comparative Proteome Analysis.

Muse cells are adult stem cells that are present in the stroma of several organs and possess an enduring capacity to cope with endogenous and exogenous genotoxic stress. In cell therapy, the peculiar biological properties of Muse cells render them a possible natural alternative to mesenchymal stromal cells (MSCs) or to in vitro-generated pluripotent stem cells (iPSCs). Indeed, some studies have proved that Muse cells can survive in adverse microenvironments, such as those present in damaged/injured tissues. We performed an evaluation of Muse cells' proteome under basic conditions and followed oxidative stress treatment in order to identify ontologies, pathways, and networks that can be related to their enduring stress capacity. We executed the same analysis on iPSCs and MSCs, as a comparison. The Muse cells are enriched in several ontologies and pathways, such as endosomal vacuolar trafficking related to stress response, ubiquitin and proteasome degradation, and reactive oxygen scavenging. In Muse cells, the protein-protein interacting network has two key nodes with a high connectivity degree and betweenness: NFKB and CRKL. The protein NFKB is an almost-ubiquitous transcription factor related to many biological processes and can also have a role in protecting cells from apoptosis during exposure to a variety of stressors. CRKL is an adaptor protein and constitutes an integral part of the stress-activated protein kinase (SAPK) pathway. The identified pathways and networks are all involved in the quality control of cell components and may explain the stress resistance of Muse cells.

Obesity induced by high-fat diet is associated with critical changes in biological and molecular functions of mesenchymal stromal cells present in visceral adipose tissue.

The mesenchymal stromal cells (MSCs) residing within the stromal component of visceral adipose tissue appear to be greatly affected by obesity, with impairment of their functions and presence of senescence. To gain further insight into these phenomena, we analyzed the changes in total proteome content and secretome of mouse MSCs after a high-fat diet (HFD) treatment compared to a normal diet (ND). In healthy conditions, MSCs are endowed with functions mainly devoted to vesicle trafficking. These cells have an immunoregulatory role, affecting leukocyte activation and migration, acute inflammation phase response, chemokine signaling, and platelet activities. They also present a robust response to stress. We identified four signaling pathways (TGF-ß, VEGFR2, HMGB1, and Leptin) that appear to govern the cells' functions. In the obese mice, MSCs showed a change in their functions. The immunoregulation shifted toward pro-inflammatory tasks with the activation of interleukin-1 pathway and of Granzyme A signaling. Moreover, the methionine degradation pathway and the processing of capped intronless pre-mRNAs may be related to the inflammation process. The signaling pathways we identified in ND MSCs were replaced by MET, WNT, and FGFR2 signal transduction, which may play a role in promoting inflammation, cancer, and aging.

Percutaneous Lower Blepharoplasty: A Novel Technique in Lower Eyelid Aesthetics.

BACKGROUND: More preservative approaches are being adopted in periorbital surgery each day. To avoid the possible complications of transcutaneous and transconjunctival blepharoplasty, the author has developed the safe and simple "Percutaneous Lower Blepharoplasty" technique, which he has been performing since 2008. The technique consists of the removal and transfer of lower orbital fat pads through 4-mm skin incisions with minimal dissection. PATIENTS AND METHODS: The study is designed as a retrospective analysis of the operations done by the author from 2008 to 2018. The author performed 297 percutaneous lower blepharoplasty operations alone or in combination with tear-trough deformity correction, pinch skin excision, and/or carbon dioxide laser application. The video of the technique is demonstrated in the supplemental digital content. RESULTS: The patients were followed for an average of 58.8 months. All the patients were satisfied with the result. There were no retractions or scleral show seen. The only complication was chemosis in 1 patient that subsided with steroid ointment. CONCLUSION: Percutaneous lower blepharoplasty is a novel technique that has the following advantages: Simple and fast, low complication rates, good cosmetic outcomes, tear trough deformity correction, better estimation of fat amount to be removed, and minimal dissection and avoidance of violation of lamellar structures. It can be performed safely alone or in combination with pinch excision and/or fractional laser resurfacing.

A 200-year reconstruction of Kocasu River (Sakarya River Basin, Turkey) streamflow derived from a tree-ring network.

The Sakarya River Basin (SRB) contains one of the most important agricultural areas for Turkey. Here, we use a network of 18 tree-ring chronologies and present a reconstruction of the mean June-July Kocasu River discharge, one of the main channels in the SRB, during the period 1803-2002 CE, and place the short period of instrumental flows (since 1953 CE) into historical context. Over the past two centuries, we found 33 dry and 28 wet events and observed the longest wet period between the years 1880 and 1920. The driest years were 1845 and 1873, and the wettest years were 1859 and 1960. Our reconstruction showed that the extreme short-term drought events that occurred in recent years were minor compared to the severity and duration of droughts that occurred previous to instrumental data. We found four pre-instrumental severe and sustained low streamflow events during the periods 1819-1834, 1840-1852, 1861-1875, and 1925-1931, during which historical records show reduced agricultural production, death, famine, plague, economic crisis, and widespread human migrations. More concerning, however, are current hydroclimate conditions in the SRB, marked by decadal-scale mean flows that dip below the long-term mean (1803-1953) in the late 1970s and have since failed to recover. With the Mediterranean region currently likely experiencing the worst drought in the past ca 1000 years due to human-induced climate change, the future outlook of water resource availability in the SRB could prove catastrophic for human and natural systems.

MASH Suite Pro: A Comprehensive Software Tool for Top-Down Proteomics.

Top-down mass spectrometry (MS)-based proteomics is arguably a disruptive technology for the comprehensive analysis of all proteoforms arising from genetic variation, alternative splicing, and posttranslational modifications (PTMs). However, the complexity of top-down high-resolution mass spectra presents a significant challenge for data analysis. In contrast to the well-developed software packages available for data analysis in bottom-up proteomics, the data analysis tools in top-down proteomics remain underdeveloped. Moreover, despite recent efforts to develop algorithms and tools for the deconvolution of top-down high-resolution mass spectra and the identification of proteins from complex mixtures, a multifunctional software platform, which allows for the identification, quantitation, and characterization of proteoforms with visual validation, is still lacking. Herein, we have developed MASH Suite Pro, a comprehensive software tool for top-down proteomics with multifaceted functionality. MASH Suite Pro is capable of processing high-resolution MS and tandem MS (MS/MS) data using two deconvolution algorithms to optimize protein identification results. In addition, MASH Suite Pro allows for the characterization of PTMs and sequence variations, as well as the relative quantitation of multiple proteoforms in different experimental conditions. The program also provides visualization components for validation and correction of the computational outputs. Furthermore, MASH Suite Pro facilitates data reporting and presentation via direct output of the graphics. Thus, MASH Suite Pro significantly simplifies and speeds up the interpretation of high-resolution top-down proteomics data by integrating tools for protein identification, quantitation, characterization, and visual validation into a customizable and user-friendly interface. We envision that MASH Suite Pro will play an integral role in advancing the burgeoning field of top-down proteomics.

Comprehensive assessment of chamber-specific and transmural heterogeneity in myofilament protein phosphorylation by top-down mass spectrometry.

The heart is characterized by a remarkable degree of heterogeneity, the basis of which is a subject of active investigation. Myofilament protein post-translational modifications (PTMs) represent a critical mechanism regulating cardiac contractility, and emerging evidence shows that pathological cardiac conditions induce contractile heterogeneity that correlates with transmural variations in the modification status of myofilament proteins. Nevertheless, whether there exists basal heterogeneity in myofilament protein PTMs in the heart remains unclear. Here we have systematically assessed chamber-specific and transmural variations in myofilament protein PTMs, specifically, the phosphorylation of cardiac troponin I (cTnI), cardiac troponin T (cTnT), tropomyosin (Tpm), and myosin light chain 2 (MLC2). We show that the phosphorylation of cTnI and αTm vary in the different chambers of the heart, whereas the phosphorylation of MLC2 and cTnT does not. In contrast, no significant transmural differences were observed in the phosphorylation of any of the myofilament proteins analyzed. These results highlight the importance of appropriate tissue sampling-particularly for studies aimed at elucidating disease mechanisms and biomarker discovery-in order to minimize potential variations arising from basal heterogeneity in myofilament PTMs in the heart.

Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation.

Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function.

Three dimensional liquid chromatography coupling ion exchange chromatography/hydrophobic interaction chromatography/reverse phase chromatography for effective protein separation in top-down proteomics.

To address the complexity of the proteome in mass spectrometry (MS)-based top-down proteomics, multidimensional liquid chromatography (MDLC) strategies that can effectively separate proteins with high resolution and automation are highly desirable. Although various MDLC methods that can effectively separate peptides from protein digests exist, very few MDLC strategies, primarily consisting of 2DLC, are available for intact protein separation, which is insufficient to address the complexity of the proteome. We recently demonstrated that hydrophobic interaction chromatography (HIC) utilizing a MS-compatible salt can provide high resolution separation of intact proteins for top-down proteomics. Herein, we have developed a novel 3DLC strategy by coupling HIC with ion exchange chromatography (IEC) and reverse phase chromatography (RPC) for intact protein separation. We demonstrated that a 3D (IEC-HIC-RPC) approach greatly outperformed the conventional 2D IEC-RPC approach. For the same IEC fraction (out of 35 fractions) from a crude HEK 293 cell lysate, a total of 640 proteins were identified in the 3D approach (corresponding to 201 nonredundant proteins) as compared to 47 in the 2D approach, whereas simply prolonging the gradients in RPC in the 2D approach only led to minimal improvement in protein separation and identifications. Therefore, this novel 3DLC method has great potential for effective separation of intact proteins to achieve deep proteome coverage in top-down proteomics.

New mass-spectrometry-compatible degradable surfactant for tissue proteomics.

Tissue proteomics is increasingly recognized for its role in biomarker discovery and disease mechanism investigation. However, protein solubility remains a significant challenge in mass spectrometry (MS)-based tissue proteomics. Conventional surfactants such as sodium dodecyl sulfate (SDS), the preferred surfactant for protein solubilization, are not compatible with MS. Herein, we have screened a library of surfactant-like compounds and discovered an MS-compatible degradable surfactant (MaSDeS) for tissue proteomics that solubilizes all categories of proteins with performance comparable to SDS. The use of MaSDeS in the tissue extraction significantly improves the total number of protein identifications from commonly used tissues, including tissue from the heart, liver, and lung. Notably, MaSDeS significantly enriches membrane proteins, which are often under-represented in proteomics studies. The acid degradable nature of MaSDeS makes it amenable for high-throughput MS-based proteomics. In addition, the thermostability of MaSDeS allows for its use in experiments requiring high temperature to facilitate protein extraction and solubilization. Furthermore, we have shown that MaSDeS outperforms the other MS-compatible surfactants in terms of overall protein solubility and the total number of identified proteins in tissue proteomics. Thus, the use of MaSDeS will greatly advance tissue proteomics and realize its potential in basic biomedical and clinical research. MaSDeS could be utilized in a variety of proteomics studies as well as general biochemical and biological experiments that employ surfactants for protein solubilization.

Top-down proteomics reveals concerted reductions in myofilament and Z-disc protein phosphorylation after acute myocardial infarction.

Heart failure (HF) is a leading cause of morbidity and mortality worldwide and is most often precipitated by myocardial infarction. However, the molecular changes driving cardiac dysfunction immediately after myocardial infarction remain poorly understood. Myofilament proteins, responsible for cardiac contraction and relaxation, play critical roles in signal reception and transduction in HF. Post-translational modifications of myofilament proteins afford a mechanism for the beat-to-beat regulation of cardiac function. Thus it is of paramount importance to gain a comprehensive understanding of post-translational modifications of myofilament proteins involved in regulating early molecular events in the post-infarcted myocardium. We have developed a novel liquid chromatography-mass spectrometry-based top-down proteomics strategy to comprehensively assess the modifications of key cardiac proteins in the myofilament subproteome extracted from a minimal amount of myocardial tissue with high reproducibility and throughput. The entire procedure, including tissue homogenization, myofilament extraction, and on-line LC/MS, takes less than three hours. Notably, enabled by this novel top-down proteomics technology, we discovered a concerted significant reduction in the phosphorylation of three crucial cardiac proteins in acutely infarcted swine myocardium: cardiac troponin I and myosin regulatory light chain of the myofilaments and, unexpectedly, enigma homolog isoform 2 (ENH2) of the Z-disc. Furthermore, top-down MS allowed us to comprehensively sequence these proteins and pinpoint their phosphorylation sites. For the first time, we have characterized the sequence of ENH2 and identified it as a phosphoprotein. ENH2 is localized at the Z-disc, which has been increasingly recognized for its role as a nodal point in cardiac signaling. Thus our proteomics discovery opens up new avenues for the investigation of concerted signaling between myofilament and Z-disc in the early molecular events that contribute to cardiac dysfunction and progression to HF.

MASH Suite: a user-friendly and versatile software interface for high-resolution mass spectrometry data interpretation and visualization.

The rapid advancements in mass spectrometry (MS) instrumentation, particularly in Fourier transform (FT) MS, have made the acquisition of high-resolution and high-accuracy mass measurements routine. However, the software tools for the interpretation of high-resolution MS data are underdeveloped. Although several algorithms for the automatic processing of high-resolution MS data are available, there is still an urgent need for a user-friendly interface with functions that allow users to visualize and validate the computational output. Therefore, we have developed MASH Suite, a user-friendly and versatile software interface for processing high-resolution MS data. MASH Suite contains a wide range of features that allow users to easily navigate through data analysis, visualize complex high-resolution MS data, and manually validate automatically processed results. Furthermore, it provides easy, fast, and reliable interpretation of top-down, middle-down, and bottom-up MS data. MASH Suite is convenient, easily operated, and freely available. It can greatly facilitate the comprehensive interpretation and validation of high-resolution MS data with high accuracy and reliability.

Top-down quantitative proteomics identified phosphorylation of cardiac troponin I as a candidate biomarker for chronic heart failure.

The rapid increase in the prevalence of chronic heart failure (CHF) worldwide underscores an urgent need to identify biomarkers for the early detection of CHF. Post-translational modifications (PTMs) are associated with many critical signaling events during disease progression and thus offer a plethora of candidate biomarkers. We have employed a top-down quantitative proteomics methodology for comprehensive assessment of PTMs in whole proteins extracted from normal and diseased tissues. We systematically analyzed 36 clinical human heart tissue samples and identified phosphorylation of cardiac troponin I (cTnI) as a candidate biomarker for CHF. The relative percentages of the total phosphorylated cTnI forms over the entire cTnI populations (%P(total)) were 56.4 ± 3.5%, 36.9 ± 1.6%, 6.1 ± 2.4%, and 1.0 ± 0.6% for postmortem hearts with normal cardiac function (n = 7), early stage of mild hypertrophy (n = 5), severe hypertrophy/dilation (n = 4), and end-stage CHF (n = 6), respectively. In fresh transplant samples, the %P(total) of cTnI from nonfailing donor (n = 4), and end-stage failing hearts (n = 10) were 49.5 ± 5.9% and 18.8 ± 2.9%, respectively. Top-down MS with electron capture dissociation unequivocally localized the altered phosphorylation sites to Ser22/23 and determined the order of phosphorylation/dephosphorylation. This study represents the first clinical application of top-down MS-based quantitative proteomics for biomarker discovery from tissues, highlighting the potential of PTMs as disease biomarkers.