Multi-omics of a pre-clinical model of diabetic cardiomyopathy reveals increased fatty acid supply impacts mitochondrial metabolic selectivity.

The incidence of type 2 diabetes (T2D) is increasing globally, with long-term implications for human health and longevity. Heart disease is the leading cause of death in T2D patients, who display an elevated risk of an acute cardiovascular event and worse outcomes following such an insult. The underlying mechanisms that predispose the diabetic heart to this poor prognosis remain to be defined. This study developed a pre-clinical model (Rattus norvegicus) that complemented caloric excess from a high-fat diet (HFD) and pancreatic ß-cell dysfunction from streptozotocin (STZ) to produce hyperglycaemia, peripheral insulin resistance, hyperlipidaemia and elevated fat mass to mimic the clinical features of T2D. Ex vivo cardiac function was assessed using Langendorff perfusion with systolic and diastolic contractile depression observed in T2D hearts. Cohorts representing untreated, individual HFD- or STZ-treatments and the combined HFD + STZ approach were used to generate ventricular samples (n = 9 per cohort) for sequential and integrated analysis of the proteome, lipidome and metabolome by liquid chromatography-tandem mass spectrometry. This study found that in T2D hearts, HFD treatment primed the metabolome, while STZ treatment was the major driver for changes in the proteome. Both treatments equally impacted the lipidome. Our data suggest that increases in ß-oxidation and early TCA cycle intermediates promoted rerouting via 2-oxaloacetate to glutamate, γ-aminobutyric acid and glutathione. Furthermore, we suggest that the T2D heart activates networks to redistribute excess acetyl-CoA towards ketogenesis and incomplete ß-oxidation through the formation of short-chain acylcarnitine species. Multi-omics provided a global and comprehensive molecular view of the diabetic heart, which distributes substrates and products from excess ß-oxidation, reduces metabolic flexibility and impairs capacity to restore high energy reservoirs needed to respond to and prevent subsequent acute cardiovascular events.

Therapeutic Inhibition of Acid-Sensing Ion Channel 1a Recovers Heart Function After Ischemia-Reperfusion Injury.

BACKGROUND: Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function. METHODS: We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell-derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents. RESULTS: Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell-derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. CONCLUSIONS: Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.

A novel phosphoproteomic landscape evoked in response to type I interferon in the brain and in glial cells.

BACKGROUND: Type I interferons (IFN-I) are key responders to central nervous system infection and injury and are also increased in common neurodegenerative diseases. Their effects are primarily mediated via transcriptional regulation of several hundred interferon-regulated genes. In addition, IFN-I activate several kinases including members of the MAPK and PI3K families. Yet, how changes to the global protein phosphoproteome contribute to the cellular response to IFN-I is unknown. METHODS: The cerebral phosphoproteome of mice with brain-targeted chronic production of the IFN-I, IFN-α, was obtained. Changes in phosphorylation were analyzed by ontology and pathway analysis and kinase enrichment predictions. These were verified by phenotypic analysis, immunohistochemistry and immunoblots. In addition, primary murine microglia and astrocytes, the brain's primary IFN-I-responding cells, were acutely treated with IFN-α and the global phosphoproteome was similarly analyzed. RESULTS: We identified widespread protein phosphorylation as a novel mechanism by which IFN-I mediate their effects. In our mouse model for IFN-I-induced neurodegeneration, protein phosphorylation, rather than the proteome, aligned with the clinical hallmarks and pathological outcome, including impaired development, motor dysfunction and seizures. In vitro experiments revealed extensive and rapid IFN-I-induced protein phosphorylation in microglia and astrocytes. Response to acute IFN-I stimulation was independent of gene expression and mediated by a small number of kinase families. The changes in the phosphoproteome affected a diverse range of cellular processes and functional analysis suggested that this response induced an immediate reactive state and prepared cells for subsequent transcriptional responses. CONCLUSIONS: Our studies reveal a hitherto unappreciated role for changes in the protein phosphorylation landscape in cellular responses to IFN-I and thus provide insights for novel diagnostic and therapeutic strategies for neurological diseases caused by IFN-I.

Proteomics Reveals Multiple Phenotypes Associated with N-linked Glycosylation in Campylobacter jejuni.

Campylobacter jejuni is a major gastrointestinal pathogen generally acquired via consumption of poorly prepared poultry. N-linked protein glycosylation encoded by the pgl gene cluster targets >80 membrane proteins and is required for both nonsymptomatic chicken colonization and full human virulence. Despite this, the biological functions of N-glycosylation remain unknown. We examined the effects of pgl gene deletion on the C. jejuni proteome using label-based liquid chromatography/tandem mass spectrometry (LC-MS/MS) and validation using data independent acquisition (DIA-SWATH-MS). We quantified 1359 proteins corresponding to ∼84% of the C. jejuni NCTC 11168 genome, and 1080 of these were validated by DIA-SWATH-MS. Deletion of the pglB oligosaccharyltransferase (ΔpglB) resulted in a significant change in abundance of 185 proteins, 137 of which were restored to their wild-type levels by reintroduction of pglB (Δaaz.batpglB::ΔpglB). Deletion of pglB was associated with significantly reduced abundances of pgl targets and increased stress-related proteins, including ClpB, GroEL, GroES, GrpE and DnaK. pglB mutants demonstrated reduced survival following temperature (4 °C and 46 °C) and osmotic (150 mm NaCl) shock and altered biofilm phenotypes compared with wild-type C. jejuni Targeted metabolomics established that pgl negative C. jejuni switched from aspartate (Asp) to proline (Pro) uptake and accumulated intracellular succinate related to proteome changes including elevated PutP/PutA (proline transport and utilization), and reduced DctA/DcuB (aspartate import and succinate export, respectively). ΔpglB chemotaxis to some substrates (Asp, glutamate, succinate and α-ketoglutarate) was reduced and associated with altered abundance of transducer-like (Tlp) proteins. Glycosylation negative C. jejuni were depleted of all respiration-associated proteins that allow the use of alternative electron acceptors under low oxygen. We demonstrate for the first time that N-glycosylation is required for a specific enzyme activity (Nap nitrate reductase) that is associated with reduced abundance of the NapAB glycoproteins. These data indicate a multifactorial role for N-glycosylation in C. jejuni physiology.

Human macrophage cathepsin B-mediated C-terminal cleavage of apolipoprotein A-I at Ser228 severely impairs antiatherogenic capacity.

Apolipoprotein A-I (apoA-I) is the major component of HDL and central to the ability of HDL to stimulate ATP-binding cassette transporter A1 (ABCA1)-dependent, antiatherogenic export of cholesterol from macrophage foam cells, a key player in the pathology of atherosclerosis. Cell-mediated modifications of apoA-I, such as chlorination, nitration, oxidation, and proteolysis, can impair its antiatherogenic function, although it is unknown whether macrophages themselves contribute to such modifications. To investigate this, human monocyte-derived macrophages (HMDMs) were incubated with human apoA-I under conditions used to induce cholesterol export. Two-dimensional gel electrophoresis and Western blot analysis identified that apoA-I is cleaved (∼20-80%) by HMDMs in a time-dependent manner, generating apoA-I of lower MW and isoelectric point. Mass spectrometry analysis identified a novel C-terminal cleavage site of apoA-I between Ser228-Phe229 Recombinant apoA-I truncated at Ser228 demonstrated profound loss of capacity to solubilize lipid and to promote ABCA1-dependent cholesterol efflux. Protease inhibitors, small interfering RNA knockdown in HMDMs, mass spectrometry analysis, and cathepsin B activity assays identified secreted cathepsin B as responsible for apoA-I cleavage at Ser228 Importantly, C-terminal cleavage of apoA-I was also detected in human carotid plaque. Cleavage at Ser228 is a novel, functionally important post-translational modification of apoA-I mediated by HMDMs that limits the antiatherogenic properties of apoA-I.-Dinnes, D. L. M., White, M. Y., Kockx, M., Traini, M., Hsieh, V., Kim, M.-J., Hou, L., Jessup, W., Rye, K.-A., Thaysen-Andersen, M., Cordwell, S. J., Kritharides, L. Human macrophage cathepsin B-mediated C-terminal cleavage of apolipoprotein A-I at Ser228 severely impairs antiatherogenic capacity.

Global analysis of myocardial peptides containing cysteines with irreversible sulfinic and sulfonic acid post-translational modifications.

Cysteine (Cys) oxidation is a crucial post-translational modification (PTM) associated with redox signaling and oxidative stress. As Cys is highly reactive to oxidants it forms a range of post-translational modifications, some that are biologically reversible (e.g. disulfides, Cys sulfenic acid) and others (Cys sulfinic [Cys-SO2H] and sulfonic [Cys-SO3H] acids) that are considered "irreversible." We developed an enrichment method to isolate Cys-SO2H/SO3H-containing peptides from complex tissue lysates that is compatible with tandem mass spectrometry (MS/MS). The acidity of these post-translational modification (pKa Cys-SO3H < 0) creates a unique charge distribution when localized on tryptic peptides at acidic pH that can be utilized for their purification. The method is based on electrostatic repulsion of Cys-SO2H/SO3H-containing peptides from cationic resins (i.e. "negative" selection) followed by "positive" selection using hydrophilic interaction liquid chromatography. Modification of strong cation exchange protocols decreased the complexity of initial flowthrough fractions by allowing for hydrophobic retention of neutral peptides. Coupling of strong cation exchange and hydrophilic interaction liquid chromatography allowed for increased enrichment of Cys-SO2H/SO3H (up to 80%) from other modified peptides. We identified 181 Cys-SO2H/SO3H sites from rat myocardial tissue subjected to physiologically relevant concentrations of H2O2 (<100 µm) or to ischemia/reperfusion (I/R) injury via Langendorff perfusion. I/R significantly increased Cys-SO2H/SO3H-modified peptides from proteins involved in energy utilization and contractility, as well as those involved in oxidative damage and repair.

Structural basis for phosphorylation and lysine acetylation cross-talk in a kinase motif associated with myocardial ischemia and cardioprotection.

Myocardial ischemia and cardioprotection by ischemic pre-conditioning induce signal networks aimed at survival or cell death if the ischemic period is prolonged. These pathways are mediated by protein post-translational modifications that are hypothesized to cross-talk with and regulate each other. Phosphopeptides and lysine-acetylated peptides were quantified in isolated rat hearts subjected to ischemia or ischemic pre-conditioning, with and without splitomicin inhibition of lysine deacetylation. We show lysine acetylation (acetyl-Lys)-dependent activation of AMP-activated protein kinase, AKT, and PKA kinases during ischemia. Phosphorylation and acetyl-Lys sites mapped onto tertiary structures were proximal in >50% of proteins investigated, yet they were mutually exclusive in 50 ischemic pre-conditioning- and/or ischemia-associated peptides containing the KXXS basophilic protein kinase consensus motif. Modifications in this motif were modeled in the C terminus of muscle-type creatine kinase. Acetyl-Lys increased proximal dephosphorylation by 10-fold. Structural analysis of modified muscle-type creatine kinase peptide variants by two-dimensional NMR revealed stabilization via a lysine-phosphate salt bridge, which was disrupted by acetyl-Lys resulting in backbone flexibility and increased phosphatase accessibility.

Secretome of transmissible Pseudomonas aeruginosa AES-1R grown in a cystic fibrosis lung-like environment.

Pseudomonas aeruginosa is the predominant cause of mortality in patients with cystic fibrosis (CF). We examined the secretome of an acute, transmissible CF P. aeruginosa (Australian epidemic strain 1-R; AES-1R) compared with laboratory-adapted PAO1. Culture supernatant proteins from rich (LB) and minimal (M9) media were compared using 2-DE and 2DLC-MS/MS, which revealed elevated abundance of PasP protease and absence of AprA protease in AES-1R. CF lung-like artificial sputum medium (ASMDM) contains serum and mucin that generally preclude proteomics of secreted proteins. ASMDM culture supernatants were subjected to 2DLC-MS/MS, which allowed the identification of 57 P. aeruginosa proteins, and qualitative spectral counting was used to estimate relative abundance. AES-1R-specific AES_7139 and PasP were more abundant in AES-1R ASMDM culture supernatants, while AprA could only be identified in PAO1. Relative quantitation was performed using selected reaction monitoring. Significantly elevated levels of PasP, LasB, chitin-binding protein (CbpD), and PA4495 were identified in AES-1R ASMDM supernatants. Quantitative PCR showed elevated pasP in AES-1R during early (18 h) ASMDM growth, while no evidence of aprA expression could be observed. Genomic screening of CF isolates revealed aes_7139 was present in all AES-1 and one pair of sequential nonepidemic isolates. Secreted proteins may be crucial in aiding CF-associated P. aeruginosa to establish infection and for adaptation to the CF lung.

Large-scale capture of peptides containing reversibly oxidized cysteines by thiol-disulfide exchange applied to the myocardial redox proteome.

Redox regulation is emerging as an important post-translational modification in cell signaling and pathogenesis. Cysteine (Cys) is the most redox active of the commonly coded amino acids and is thus an important target for redox-based modifications. Reactions that oxidize the Cys sulfur atom to low oxidation states (e.g., disulfide) are reversible, while further reactions to higher oxidation states (e.g., sulfonic acid) may be irreversible under biological conditions. Reversible modifications are particularly interesting as they mediate redox signaling and regulation of proteins under physiological conditions and during adaptation to oxidant stress. An enrichment method that relied on rapid and specific alkylation of free Cys, followed by thiol-based reduction and resin capture by thiol-disulfide exchange chemistry was applied to isolate reversibly modified Cys-containing peptides. Chromatographic conditions were optimized to provide increased specificity by removal of noncovalent interactions. The technique was highly efficient, based on near equimolar reactions with the resin, reproducible and linear for peptide elution, as quantified by label-free mass spectrometry. The method was applied to a complex protein lysate generated from rat myocardial tissue and 6559 unique Cys-containing peptides from 2694 proteins were identified. Comparison with the rat database and previous studies showed effective enrichment of proteins modified by S-nitrosylation, disulfide formation, and Cys-sulfenic acid. Analysis of amino acid sequence features indicated a preference for acidic residues and increased hydrophilicity in the regions immediately up- or downstream of the reactive Cys. This technique is ideally suited for the enrichment and profiling of reversible Cys modifications on a proteome-wide scale.

Parallel proteomics to improve coverage and confidence in the partially annotated Oryctolagus cuniculus mitochondrial proteome.

The ability to decipher the dynamic protein component of any system is determined by the inherent limitations of the technologies used, the complexity of the sample, and the existence of an annotated genome. In the absence of an annotated genome, large-scale proteomic investigations can be technically difficult. Yet the functional and biological species differences across animal models can lead to selection of partially or nonannotated organisms over those with an annotated genome. The outweighing of biology over technology leads us to investigate the degree to which a parallel approach can facilitate proteome coverage in the absence of complete genome annotation. When studying species without complete genome annotation, a particular challenge is how to ensure high proteome coverage while meeting the bioinformatic stringencies of high-throughput proteomics. A protein inventory of Oryctolagus cuniculus mitochondria was created by overlapping "protein-centric" and "peptide-centric" one-dimensional and two-dimensional liquid chromatography strategies; with additional partitioning into membrane-enriched and soluble fractions. With the use of these five parallel approaches, 2934 unique peptides were identified, corresponding to 558 nonredundant protein groups. 230 of these proteins (41%) were identified by only a single technical approach, confirming the need for parallel techniques to improve annotation. To determine the extent of coverage, a side-by-side comparison with human and mouse cardiomyocyte mitochondrial studies was performed. A nonredundant list of 995 discrete proteins was compiled, of which 244 (25%) were common across species. The current investigation identified 142 unique protein groups, the majority of which were detected here by only one technical approach, in particular peptide- and protein-centric two-dimensional liquid chromatography. Although no single approach achieved more than 40% coverage, the combination of three approaches (protein- and peptide-centric two-dimensional liquid chromatography and subfractionation) contributed 96% of all identifications. Parallel techniques ensured minimal false discovery, and reduced single peptide-based identifications while maximizing sequence coverage in the absence of the annotated rabbit proteome.

Quantitative N-linked glycoproteomics of myocardial ischemia and reperfusion injury reveals early remodeling in the extracellular environment.

Extracellular and cell surface proteins are generally modified with N-linked glycans and glycopeptide enrichment is an attractive tool to analyze these proteins. The role of N-linked glycoproteins in cardiovascular disease, particularly ischemia and reperfusion injury, is poorly understood. Observation of glycopeptides by mass spectrometry is challenging due to the presence of abundant, nonglycosylated analytes, and robust methods for purification are essential. We employed digestion with multiple proteases to increase glycoproteome coverage coupled with parallel glycopeptide enrichments using hydrazide capture, titanium dioxide, and hydrophilic interaction liquid chromatography with and without an ion-pairing agent. Glycosylated peptides were treated with PNGase F and analyzed by liquid chromatography-MS/MS. This allowed the identification of 1556 nonredundant N-linked glycosylation sites, representing 972 protein groups from ex vivo rat left ventricular myocardium. False positive "glycosylations" were observed on 44 peptides containing a deamidated Asn-Asp in the N-linked sequon by analysis of samples without PNGase F treatment. We used quantitation via isobaric tags for relative and absolute quantitation (iTRAQ) and validation with dimethyl labeling to analyze changes in glycoproteins from tissue following prolonged ischemia and reperfusion (40 mins ischemia and 20 mins reperfusion) indicative of myocardial infarction. The iTRAQ approach revealed 80 of 437 glycopeptides with altered abundance, while dimethyl labeling confirmed 46 of these and revealed an additional 62 significant changes. These were mainly from predicted extracellular matrix and basement membrane proteins that are implicated in cardiac remodeling. Analysis of N-glycans released from myocardial proteins suggest that the observed changes were not due to significant alterations in N-glycan structures. Altered proteins included the collagen-laminin-integrin complexes and collagen assembly enzymes, cadherins, mast cell proteases, proliferation-associated secreted protein acidic and rich in cysteine, and microfibril-associated proteins. The data suggest that cardiac remodeling is initiated earlier during reperfusion than previously hypothesized.

Reduction-cleavable desferrioxamine B pulldown system enriches Ni(ii)-superoxide dismutase from a Streptomyces proteome.

Two resins with the hydroxamic acid siderophore desferrioxamine B (DFOB) immobilised as a free ligand or its Fe(iii) complex were prepared to screen the Streptomyces pilosus proteome for proteins involved in siderophore-mediated Fe(iii) uptake. The resin design included a disulfide bond to enable the release of bound proteins under mild reducing conditions. Proteomics analysis of the bound fractions did not identify proteins associated with siderophore-mediated Fe(iii) uptake, but identified nickel superoxide dismutase (NiSOD), which was enriched on the apo-DFOB-resin but not the Fe(iii)-DFOB-resin or the control resin. While DFOB is unable to sequester Fe(iii) from sites deeply buried in metalloproteins, the coordinatively unsaturated Ni(ii) ion in NiSOD is present in a surface-exposed loop region at the N-terminus, which might enable partial chelation. The results were consistent with the notion that the apo-DFOB-resin formed a ternary complex with NiSOD, which was not possible for either the coordinatively saturated Fe(iii)-DFOB-resin or the non-coordinating control resin systems. In support, ESI-TOF-MS measurements from a solution of a model Ni(ii)-SOD peptide and DFOB showed signals that correlated with a ternary Ni(ii)-SOD peptide-DFOB complex. Although any biological implications of a DFOB-NiSOD complex are unclear, the work shows that the metal coordination properties of siderophores might influence an array of metal-dependent biological processes beyond those established in iron uptake.

Release of tissue-specific proteins into coronary perfusate as a model for biomarker discovery in myocardial ischemia/reperfusion injury.

Diagnosis of acute coronary syndromes is based on protein biomarkers, such as the cardiac troponins (cTnI/cTnT) and creatine kinase (CK-MB) that are released into the circulation. Biomarker discovery is focused on identifying very low abundance tissue-derived analytes from within albumin-rich plasma, in which the wide dynamic range of the native protein complement hinders classical proteomic investigations. We employed an ex vivo rabbit model of myocardial ischemia/reperfusion (I/R) injury using Langendorff buffer perfusion. Nonrecirculating perfusate was collected over a temporal profile of 60 min reperfusion following brief, reversible ischemia (15 min; 15I/60R) for comparison with irreversible I/R (60I/60R). Perfusate proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry (MS), revealing 26 tissue-specific proteins released during reperfusion post-15I. Proteins released during irreversible I/R (60I/60R) were profiled using gel-based (2-DE and one-dimensional gel electrophoresis coupled to liquid chromatography and tandem mass spectrometry; geLC-MS) and gel-free (LC-MS/MS) methods. A total of 192 tissue-specific proteins were identified during reperfusion post-60I. Identified proteins included those previously associated with I/R (myoglobin, CK-MB, cTnI, and cTnT), in addition to examples currently under investigation in large cohort studies (heart-type fatty acid binding protein; FABPH). The postischemic release profile of a novel cardiac-specific protein, cysteine and glycine-rich protein 3 (Csrp3; cardiac LIM domain protein) was validated by Western blot analysis. We also identified Csrp3 in serum from 6 of 8 patients postreperfusion following acute myocardial infarction. These studies indicate that animal modeling of biomarker release using ex vivo buffer perfused tissue to limit the presence of obfuscating plasma proteins may identify candidates for further study in humans.

A Global Profile of Reversible and Irreversible Cysteine Redox Post-Translational Modifications During Myocardial Ischemia/Reperfusion Injury and Antioxidant Intervention.

Aims: Cysteine (Cys) is a major target for redox post-translational modifications (PTMs) that occur in response to changes in the cellular redox environment. We describe multiplexed, peptide-based enrichment and quantitative mass spectrometry (MS) applied to globally profile reversible redox Cys PTM in rat hearts during ischemia/reperfusion (I/R) in the presence or absence of an aminothiol antioxidant, N-2-mercaptopropionylglycine (MPG). Parallel fractionation also allowed identification of irreversibly oxidized Cys peptides (Cys-SO2H/SO3H). Results: We identified 4505 reversibly oxidized Cys peptides of which 1372 were significantly regulated by ischemia and/or I/R. An additional 219 peptides (247 sites) contained Cys-SO2H/Cys-SO3H modifications, and these were predominantly identified from hearts subjected to I/R (n = 168 peptides). Parallel reaction monitoring MS (PRM-MS) enabled relative quantitation of 34 irreversibly oxidized Cys peptides. MPG attenuated a large cluster of I/R-associated reversibly oxidized Cys peptides and irreversible Cys oxidation to less than nonischemic controls (n = 24 and 34 peptides, respectively). PRM-MS showed that Cys sites oxidized during ischemia and/or I/R and "protected" by MPG were largely mitochondrial, and were associated with antioxidant functions (peroxiredoxins 5 and 6) and metabolic processes, including glycolysis. Metabolomics revealed I/R induced changes in glycolytic intermediates that were reversed in the presence of MPG, which were consistent with irreversible PTM of triose phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), altered GAPDH enzyme activity, and reduced I/R glycolytic payoff as evidenced by adenosine triphosphate and NADH levels. Innovation: Novel enrichment and PRM-MS approaches developed here enabled large-scale relative quantitation of Cys redox sites modified by reversible and irreversible PTM during I/R and antioxidant remediation. Conclusions: Cys sites identified here are targets of reactive oxygen species that can contribute to protein dysfunction and the pathogenesis of I/R.

The role of proteomics in clinical cardiovascular biomarker discovery.

Cardiovascular disease remains the most common cause of death in the developed world and is predicted by the World Health Organization to kill approximately 20 million people worldwide each year until at least 2015. In light of these figures, work on producing superior tools for clinical use in the cardiovascular field is intensive. As proteins are the primary effectors of cellular function, a significant majority of this work focuses on the role of proteins in the cardiovascular system in physiological and pathological states in order to outline both mechanisms and markers of disease. One of the most effective ways to investigate these on a global basis is through proteomic analysis, which allows for broad spectrum screening of cellular protein or peptide complements during cardiovascular pathogenesis. Furthermore, specific technologies are now available to screen animal model or human blood samples for novel, improved markers of chronic disease states, such as atherosclerosis or for earlier indicators of acute myocardial stress, including ischemia/reperfusion injury and heart failure. This review summarizes current literature on the key aspects of proteomics and peptidomics related to clinical cardiovascular science.

Assessment of albumin removal from an immunoaffinity spin column: critical implications for proteomic examination of the albuminome and albumin-depleted samples.

High abundance proteins in serum and plasma (e.g., albumin) are routinely removed during proteomic sample processing as they can mask lower abundance proteins and peptides of biological/clinical interest. A common method of albumin depletion is based on immunoaffinity capture, and many immunoaffinity devices are designed for multiple uses. In this case, it is critical that the albumin captured on the affinity matrix is stripped from the column prior to regeneration of the matrix and processing of subsequent samples, to ensure no carryover and that maximal binding sites are available for subsequent samples. The current study examines the ability of a manufacturer's protocol to remove the proteins and peptides captured by an immunoaffinity spin column. The data presented in the current work illustrate the difficulty in completely removing albumin from the immunoaffinity device, and consequently, may explain the variability and decreased efficiency shown for this device in previous studies. In summary, the current data present important considerations for the implementation of multiple-use immunoaffinity devices for processing subsequent clinical samples in a proteomic workflow.

Alterations to the protein profile of bladder carcinoma cell lines induced by plant extract MINA-05 in vitro.

Bladder cancer (BLCa) is a severe urological cancer of both men and women that commonly recurs and once invasive, is difficult to treat. MINA-05 (CK Life Sciences Int'l, Hong Kong) is a derivative of complex botanical extracts, shown to reduce cellular proliferation of bladder and prostate carcinomas. We tested the effects of MINA-05 against human BLCa cell sublines, B8, B8-RSP-GCK, B8-RSP-LN and C3, from a transitional cell carcinoma, grade IV, to determine the molecular targets of treatment by observing the cellular protein profile. Cells were acclimatised for 48 h then treated for 72 h with concentrations of MINA-05 reflecting 1/2 IC(50), IC(50) and 2 x IC(50) (n = 3) or with vehicle, (0.5% DMSO). Dose-dependant changes in protein abundance were detected and characterised using 2-dimensional electrophoresis and MS. We identified 10 proteins that underwent changes in abundance, pI and/or molecular mass in response to treatment. MINA-05 was shown to influence proteins across numerous functional classes including cytoskeletal proteins, energy metabolism proteins, protein degradation proteins and tumour suppressors, suggesting a global impact on these cell lines. This study implies that the ability of MINA-05 to retard cellular proliferation is attributed to its ability to alter cell cycling, metabolism, protein degradation and the cancer cell environment.

Cardiovascular Initiative of the Human Proteome Organisation, 5th Workshop October 2007, Seoul, Korea.

The Cardiovascular Initiative (CVI) of the Human Proteome Organisation (HUPO) held its fifth workshop prior to the Sixth Annual HUPO World Congress in Seoul, Korea in October 2007. The objectives of this report are as follows: to trace the (relatively brief) history of the CVI for those who may not be acquainted with it; to highlight lectures given by members of the CVI during this Workshop; and to make the community aware of the aims of this Initiative, including collaborative projects currently under consideration.

Statistical analysis of image data provided by two-dimensional gel electrophoresis for discovery proteomics.

Standardized methods for the solubilization of proteins prior to proteomics analyses incorporating two-dimensional gel electrophoresis (2-DE) are essential for providing reproducible data that can be subjected to rigorous statistical interrogation for comparative studies investigating disease-genesis. In this chapter, we discuss the imaging and image analysis of proteins separated by 2-DE, in the context of determining protein abundance alterations related to a change in biochemical or biophysical conditions. We then describe the principles behind 2-DE gel statistical analysis, including subtraction of background noise, spot detection, gel matching, spot quantitation for data comparison, and statistical requirements to create meaningful gel data sets. We also emphasize the need to develop reproducible and robust protocols for protein sample preparation and 2-DE itself.