Emerging metabolomic tools have created the opportunity to establish metabolic signatures of myocardial injury. We applied a mass spectrometry-based metabolite profiling platform to 36 patients ...undergoing alcohol septal ablation treatment for hypertrophic obstructive cardiomyopathy, a human model of planned myocardial infarction (PMI). Serial blood samples were obtained before and at various intervals after PMI, with patients undergoing elective diagnostic coronary angiography and patients with spontaneous myocardial infarction (SMI) serving as negative and positive controls, respectively. We identified changes in circulating levels of metabolites participating in pyrimidine metabolism, the tricarboxylic acid cycle and its upstream contributors, and the pentose phosphate pathway. Alterations in levels of multiple metabolites were detected as early as 10 minutes after PMI in an initial derivation group and were validated in a second, independent group of PMI patients. A PMI-derived metabolic signature consisting of aconitic acid, hypoxanthine, trimethylamine N-oxide, and threonine differentiated patients with SMI from those undergoing diagnostic coronary angiography with high accuracy, and coronary sinus sampling distinguished cardiac-derived from peripheral metabolic changes. Our results identify a role for metabolic profiling in the early detection of myocardial injury and suggest that similar approaches may be used for detection or prediction of other disease states.
Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently ...lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho‐signaling in drug‐treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy.
SYNOPSIS
SigPath is a targeted, quantitative mass spectrometry assay that measures 284 phosphosites spanning 200 phosphoproteins with high throughput and quantitative precision across a broad swath of signaling biology of known interest.
The value of the assay is demonstrated by application to drug‐treated cancer cell lines, breast cancer preclinical models and human medulloblastoma tumors.
Large numbers of differentially regulated phosphosites newly associated with disease models and human tumors are identified.
The results highlight the potential use of SigPath to nominate mechanistic hypotheses regarding oncogenesis, response and resistance to therapy.
SigPath is a targeted, quantitative mass spectrometry assay that measures 284 phosphosites spanning 200 phosphoproteins with high throughput and quantitative precision across a broad swath of signaling biology of known interest.
Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS with unknown cause. Proteins with different abundance in the cerebrospinal fluid (CSF) from relapsing‐remitting MS (RRMS) patients ...and neurological controls could give novel insight to the MS pathogenesis and be used to improve diagnosis, predict prognosis and disease course, and guide in therapy decisions. We combined iTRAQ labeling and Orbitrap mass spectrometry to discover proteins with different CSF abundance between six RRMS patients and 18 neurological disease controls. From 777 quantified proteins seven were selected as biomarker candidates, namely chitinase‐3‐like protein 1, secretogranin‐1 (Sg1), cerebellin‐1, neuroserpin, cell surface glycoprotein MUC18, testican‐2 and glutamate receptor 4. An independent sample set of 13 early‐MS patients, 13 RRMS patients and 13 neurological controls was used in a multiple reaction monitoring verification study. We found the intracellular calcium binding protein Sg1 to be increased in early‐MS patients compared to RRMS and neurological controls. Sg1 should be included in further studies to elucidate its role in the early phases of MS pathogenesis and its potential as a biomarker for this disease.
Proteomic characterization of blood plasma is of central importance to clinical proteomics and particularly to biomarker discovery studies. The vast dynamic range and high complexity of the plasma ...proteome have, however, proven to be serious challenges and have often led to unacceptable tradeoffs between depth of coverage and sample throughput. We present an optimized sample-processing pipeline for analysis of the human plasma proteome that provides greatly increased depth of detection, improved quantitative precision and much higher sample analysis throughput as compared with prior methods. The process includes abundant protein depletion, isobaric labeling at the peptide level for multiplexed relative quantification and ultra-high-performance liquid chromatography coupled to accurate-mass, high-resolution tandem mass spectrometry analysis of peptides fractionated off-line by basic pH reversed-phase (bRP) chromatography. The overall reproducibility of the process, including immunoaffinity depletion, is high, with a process replicate coefficient of variation (CV) of <12%. Using isobaric tags for relative and absolute quantitation (iTRAQ) 4-plex, >4,500 proteins are detected and quantified per patient sample on average, with two or more peptides per protein and starting from as little as 200 μl of plasma. The approach can be multiplexed up to 10-plex using tandem mass tags (TMT) reagents, further increasing throughput, albeit with some decrease in the number of proteins quantified. In addition, we provide a rapid protocol for analysis of nonfractionated depleted plasma samples analyzed in 10-plex. This provides ∼600 quantified proteins for each of the ten samples in ∼5 h of instrument time.
Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem ...and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation.
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•Molecular lesions underlying DBA reduce ribosome levels in hematopoietic cells•Ribosome composition remains constant in cells with DBA-associated lesions•Reduced ribosome levels selectively impair translation of a subset of mRNAs•Translational perturbations in DBA impair lineage-commitment in HSPCs
A global reduction in ribosome levels in Diamond-Blackfan anemia profoundly alters translation of a select subset of transcripts, thereby impeding erythroid lineage commitment.
Background
Exercise training promotes metabolic wellness, prevents many disorders, and has therapeutic benefits, such as in cardiovascular disease and non‐alcoholic fatty liver disease. While the ...benefits of exercise are accepted, the underlying molecular mechanisms are not well understood.
Protein changes are critical in the regulation of signaling pathways and enzymatic activities associated with cellular and metabolic functions in response to exercise training. Mass spectrometry (MS)‐based proteomic technologies enable extensive profiling and quantification of proteins and protein post‐translational modifications (PTMs). Proteomic approaches have been primarily applied to the study of skeletal muscle adaption to exercise. However, few studies have leveraged these novel technologies to study key organs that adapt and benefit from exercise, such as heart and liver.
Purpose
To provide an integrative view of the molecular response to endurance exercise training by profiling the proteome, phosphoproteome, and acetylome of rat liver and heart tissues.
Method
Male and female 6‐month old rats were subjected to a training regime that consists of 30 minutes of treadmill running (≥ 70% VO2 max) and were sacrificed after 1, 2, 4, and 8 weeks of training. Liver and heart tissue samples were processed for quantitative proteomic analysis using isobaric chemical mass‐tag labeling (TMT) and fractionation of peptides for deep‐scale global proteome analysis. In addition, phosphopeptides and acetyl peptides were enriched and analyzed by MS for quantification of the phosphoproteome and acetylome, respectively.
Results
We quantified more than 9000 proteins, 35000 phospho‐, and 7000 acetyl‐peptides in heart and liver from rats undergoing aerobic exercise training. Pathway analysis of the temporal phosphoproteome responses revealed rewiring of multiple signaling pathways, such as insulin response networks (Figure 1). We observed substantial differential protein acetylation in both liver and heart. Mitochondria proteins in the liver showed a robust increase in acetylation, including those in pathways involved in metabolic disorders such as branched chain amino acid metabolism. Temporal analysis of acetylation revealed an early peak in deacetylation of heart structural proteins, suggesting early adaptation of heart fibers in response to exercise training. In contrast, changes in metabolic and peroxisomal pathway occurred at later time points of training.
Conclusion
This study reveals novel molecular pathways with potential roles in exercise adaptation, including the extensive regulation of liver and heart proteins through acetylation.
Exercise provides a robust physiological stimulus that evokes cross-talk among multiple tissues that when repeated regularly (i.e., training) improves physiological capacity, benefits numerous organ ...systems, and decreases the risk for premature mortality. However, a gap remains in identifying the detailed molecular signals induced by exercise that benefits health and prevents disease. The Molecular Transducers of Physical Activity Consortium (MoTrPAC) was established to address this gap and generate a molecular map of exercise. Preclinical and clinical studies will examine the systemic effects of endurance and resistance exercise across a range of ages and fitness levels by molecular probing of multiple tissues before and after acute and chronic exercise. From this multi-omic and bioinformatic analysis, a molecular map of exercise will be established. Altogether, MoTrPAC will provide a public database that is expected to enhance our understanding of the health benefits of exercise and to provide insight into how physical activity mitigates disease.
The Molecular Transducers of Physical Activity Consortium aims to create a comprehensive, integrative multi-omic map of the effects of exercise across tissues in rodents and healthy people.
BACKGROUND:Single-stranded DNA aptamers are oligonucleotides of ≈50 base pairs in length selected for their ability to bind proteins with high specificity and affinity. Emerging DNA aptamer-based ...technologies may address limitations of existing proteomic techniques, including low sample throughput, which have hindered proteomic analyses of large cohorts.
METHODS:To identify early biomarkers of myocardial injury, we applied an aptamer-based proteomic platform that measures 1129 proteins to a clinically relevant perturbational model of planned myocardial infarction (PMI), patients undergoing septal ablation for hypertrophic cardiomyopathy. Blood samples were obtained before and at 10 and 60 minutes after PMI, and protein changes were assessed by repeated-measures analysis of variance. The generalizability of our PMI findings was evaluated in a spontaneous myocardial infarction cohort (Wilcoxon rank-sum). We then tested the platform’s ability to detect associations between proteins and Framingham Risk Score components in the Framingham Heart Study, performing regression analyses for each protein versus each clinical trait.
RESULTS:We found 217 proteins that significantly changed in the peripheral vein blood after PMI in a derivation cohort (n=15; P<5.70E-5). Seventy-nine of these proteins were validated in an independent PMI cohort (n=15; P<2.30E-4); >85% were directionally consistent and reached nominal significance. We detected many protein changes that are novel in the context of myocardial injury, including Dickkopf-related protein 4, a WNT pathway inhibitor (peak increase 124%, P=1.29E-15) and cripto, a growth factor important in cardiac development (peak increase 64%, P=1.74E-4). Among the 40 validated proteins that increased within 1 hour after PMI, 23 were also elevated in patients with spontaneous myocardial infarction (n=46; P<0.05). Framingham Heart Study analyses revealed 156 significant protein associations with the Framingham Risk Score (n=899), including aminoacylase 1 (β=0.3386, P=2.54E-22) and trigger factor 2 (β=0.2846, P=5.71E-17). Furthermore, we developed a novel workflow integrating DNA-based immunoaffinity with mass spectrometry to analytically validate aptamer specificity.
CONCLUSIONS:Our results highlight an emerging proteomics tool capable of profiling >1000 low-abundance analytes with high sensitivity and high precision, applicable both to well-phenotyped perturbational studies and large human cohorts, as well.
Abstract BACKGROUND Diffuse midline gliomas (DMGs) are universally fatal pediatric tumors with a median survival of less than one year. Forkhead Box R2 (FOXR2) is a forkhead family TF that we have ...found to be aberrantly expressed in DMGs. FOXR2 expression is sufficient to enhance glioma formation, and we have shown that regulation of FOXR2 occurs through a previously unrecognized epigenetic mechanism. FOXR2 is necessary for proliferation in FOXR2-expressing DMGs. It is also a potent oncogene, sufficient to enhance cell proliferation and in vivo tumor formation. However, the downstream mediators of FOXR2-mediated gliomagenesis are poorly understood. METHODS We applied an integrative approach using proteomics, transcriptomics, in vitro cancer models, and in vivo in utero electroporation mouse models to systematically evaluate how FOXR2 mediates gliomagenesis. RESULTS We have found that FOXR2 is aberrantly activated in DMGs. FOXR2 enhances MYC protein stability, even in the presence of cycloheximide. However, FOXR2 also exerts its oncogenic effects through MYC independent functions. FOXR2 is highly enriched at E26-transformation (ETS) motifs and specifically activates ETS transcriptional gene sets. To identify FOXR2 protein-protein interactions, we performed immunoprecipitation and mass spectrometry to identify FOXR2 protein interactors. Moreover, we have performed both proteomic and phospho-proteomic analyses of FOXR2-expressing human neural stem cells. These proteomic studies have allowed us to identify proteins, phospho-sites, and signaling pathways that are highly enriched in FOXR2-expressing cells. These pathways could be potential therapeutic targets in FOXR2-expressing DMGs. CONCLUSIONS Taken together, this study elucidates how FOXR2 protein interactors mediate oncogenesis in FOXR2-expressing diffuse midline gliomas.
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