Showing papers by "Jennifer E. Van Eyk published in 2007"

Clinical proteomics: A need to define the field and to begin to set adequate standards.

Abstract: The aim of this manuscript is to initiate a constructive discussion about the definition of clinical proteomics, study requirements, pitfalls and (potential) use. Furthermore, we hope to stimulate proposals for the optimal use of future opportunities and seek unification of the approaches in clinical proteomic studies. We have outlined our collective views about the basic principles that should be considered in clinical proteomic studies, including sample selection, choice of technology and appropriate quality control, and the need for collaborative interdisciplinary efforts involving clinicians and scientists. Furthermore, we propose guidelines for the critical aspects that should be included in published reports. Our hope is that, as a result of stimulating discussion, a consensus will be reached amongst the scientific community leading to guidelines for the studies, similar to those already published for mass spectrometric sequencing data. We contend that clinical proteomics is not just a collection of studies dealing with analysis of clinical samples. Rather, the essence of clinical proteomics should be to address clinically relevant questions and to improve the state-of-the-art, both in diagnosis and in therapy of diseases.

Investigation of an albumin-enriched fraction of human serum and its albuminome.

Abstract: The removal of albumin and other high abundance proteins is a routine first step in the analysis of serum and plasma proteomes. However, as albumin can bind proteins and peptides, there is a universal concern as to how the serum proteome is changed by the removal of albumin. To address this concern, the current study was designed to identify proteins and peptides removed from the serum during albumin depletion; to determine which of these are bound to albumin (rather than copurified) and whether the bound proteins are intact proteins or peptide fragments. Sequential, independent analyses including both anti-albumin antibody (anti-HSA) affinity chromatography and SEC were used to isolate albumin-bound proteins. RP-HPLC and 1-D SDS-PAGE were then used to further separate the proteins prior to identification by MS/MS. Finally, whole protein molecular weight (MW) MS measurements coupled with protein coverage obtained by MS were combined to assess whether the bound proteins were intact or peptide fragments. Combining the results from multiple approaches, 35 proteins, of which 24 are intact, were found to be associated with albumin, and they include both known high and low abundance proteins.

Subfractionation of heart tissue: the "in sequence" myofilament protein extraction of myocardial tissue.

Abstract: Proteomic analysis of heart tissue is complicated by the large dynamic range of its proteins. The most abundant proteins are the myofilament proteins, which comprise the contractile apparatus. This chapter describes a protocol for fractionation of heart tissue that extracts the myofilament proteins into a separate sample fraction, allowing analysis of lower-abundance proteins. Importantly, this is performed in a manner that is compatible with two-dimensional electrophoresis and high-performance liquid chromatography, two of main technologies of proteomics. The method produces three fractions based on solubility at different pHs: (1) cytoplasmic-enriched extract (neutral pH), (2) myofilament-enriched extract (acidic pH), and (3) membrane protein-enriched pellet. Fractionation of heart tissue in this manner provides the basis for in-depth proteomic analysis.

A rapid, economical, and reproducible method for human serum delipidation and albumin and IgG removal for proteomic analysis.

Abstract: Serum is a readily available source for diagnostic assays, but the identification of disease-specific serum biomarkers has been impeded by the dominance of human serum albumin (HSA) and immunoglobulin G (IgG) in the serum proteome. Therefore, in order to observe lower-abundance serum proteins, removal or depletion of at least these two proteins is required. However, the depletion method needs to be inexpensive and reproducible. We describe such a protocol that combines delipidation by centrifugation, IgG removal with Protein G Sepharose, and HSA depletion with sodium chloride/ethanol precipitation. The protocol is streamlined to increase reproducibility and is compatible with many proteomic platforms, including two-dimensional gel electrophoresis, and high-performance liquid chromatography either offline or coupled online with a mass spectrometer. The reproducible depletion of lipids, IgG, and HSA permits a higher load of the remaining serum proteins, facilitating the identification of disease biomarkers.

Proteomics of the aqueous humor in healthy New Zealand rabbits.

Abstract: There are several physiological roles postulated for aqueous humor, a liquid located in the anterior and posterior chamber of the eye, such as maintenance of the intraocular pressure, provision of nutrients, and removal of metabolic waste from neighboring tissues and provision of an immune response and protection during inflammation and infection. To link these function to specific or classes of proteins, identification of the aqueous humor proteome is essential. Aqueous humor obtained from healthy New Zealand white rabbits was analyzed using three synergistic protein separation methods: 1-D gel electrophoresis, 2-DE, and 1-DLC (RPLC) prior to protein identification by MS. As each of these separation methods separates intact proteins based on different physical properties (pIs, molecular weights, hydrophobicity, solubility, etc.) the proteome coverage is expanded. This was confirmed, since overlap between all three separation technologies was only about 8.2% with many proteins found uniquely by a single method. Although the most dominant protein presented in normal aqueous humor is albumin, by using this extensive separation/MS strategy, additional proteins were identified in total amount of 98 nonredundant proteins (plus an additional ten proteins for consideration). This expands the current protein identifications by approximately 65%. The aqueous humor proteome comprises a specific selection of cellular and plasma based proteins and can almost exclusively be divided into four functional groups: cell-cell interactions/wound healing, proteases and protease inhibitors, antioxidant protection, and antibacterial/anti-inflammatory proteins.

Cardiovascular proteomics: past, present, and future.

Abstract: With cardiovascular (CV)-related disorders accounting for the highest mortality rates in the world, affecting the quantity and quality of life of patients and creating an economic burden of prolonged therapeutic intervention, there is great significance in understanding the cellular and molecular alterations that influence the progression of these pathologies. The cellular genotype is regulated by the DNA component, whilst the cellular phenotype is influenced by the protein complement. By improving the understanding of the molecular mechanisms that influence the protein profile, the pathologies that influence the intrinsic functions of the CV system may be detected earlier or managed more efficiently. This is achievable with technologies encompassed by 'proteomics.' Proteomic investigations of CV diseases, including dilated cardiomyopathy (DCM), atherosclerosis, and ischemia/reperfusion (I/R) injury, have identified candidate proteins altered with the pathologic states, complementing past biochemical and physiologic observations. Whilst proteomics is still a relatively new discipline to be applied to the basic scientific investigation of CV diseases, it is emerging as a technique to screen for potential biomarkers in both tissues/cells and biologic fluids (biofluids), as well as to identify the targets of existing therapeutics. By enabling the separation of complex mixtures over numerous dimensions, exploiting the intrinsic properties of proteins, including charge state, molecular mass, and hydrophobicity, in addition to cellular location, the discrete alterations within the cell may be resolved. Proteomics has shown alterations to myofilament proteins including troponin I and myosin light chain, correlating with the reduction in contractility in the myocardium from DCM and I/R. The diverse cell types that coalesce to induce atherosclerotic plaque formation have been investigated both collectively and individually to elucidate the influence of the modifications to single cell types on the developing plaque as a whole. Proteomics has also been used to observe changes to biofluids occurring with these pathologies, a new potential link between basic science and clinical applications. The development of CV proteomics has helped to identify a number of possible protein candidates, and offers the potential to treat and diagnose CV disease more effectively in the future.

Clinical proteomics : from diagnosis to therapy

Abstract: Editors Overview PART 1: TECHNOLOGIES Pre-analytical issues in clinical proteomic studies Protein separation by 2DE Protein separation : liquid chromatography HPLC in protein discovery IEF analysis of peptides for biomarkers analysis Capillary electrophoresis separations for clinical proteomics Quantitative proteomics using nanoLC with high accuracy mass spectrometry Antibody microarrays for protein and glycan detection Biomarker Identification: The Role of Experimental Design, Statistics, and Data Sharing PART 2: CANCER Applications of stable isotope tagging based quantitive proteomics in cancer research 2-D liquid seperations, protein microarrays and mass spectrometry in comprehensive analysis of PTM and biomarker discovery Development and Use of Reversed-Phase Protein Microarrays for Clinical Applications CDK1 and cancer: usefulness of proteomic approaches in assesment of the molecular mechanisms and efficacy of novel therapeutics PART 3: CARDIOVASCULAR DISEASE Diagnostic markers for monitoring heart transplant rejection The study of microheterogeneity in human plasma proteins: applications to acute myocardial infarction Discovery of biomarkers for cardiovascular diseases Development of biomarker Developfment Pipeline: Search for Myocardial Ischemia Biomarkers Albuminome as a tool for biomarker discovery PART 4: VASCULAR DISEASES Application of Metabolomics to Cardiovascular Biomarker and Pathway Discovery Urinary biomarkers in diabetic nephropathy and other glomerular diseases Pulmonary proteomics Proteomics providing insights into major psychiatric disorders PART 6: BIOMARKERS Redox Proteomics Analysis of Oxidative Modified Brain Proteins in Alzheimer's Disease and Mild Cognitive Impairment: Insights into the Progression of This Dementing Disorder Toxicoproteomics: correlating tissue and serum proteins in liver injuries Biomarkers for renal disease and uraemic toxins HIV and other viral screens PART 6: AUTOANTIBODIES AND SIGNATURE BIOMARKERS Application of Fungal Cyclic Peptides and Metabolites Microarray approaches to autoantibody profiling Identification of tumor antigen-directed autoantibodies PART 7: FUTURE Antibody and Reverse Capture Protein Microarrays Use of Antibody Microarrays in the Analysis of Inflammation, Autoimmunity, Viral Infection, and Cancer Metastases The Future: Translation from Discovery to the Clinic - Roles of HUPO and industry in biomarker discovery

Detection of Venous Thromboembolism by Proteomic Serum Biomarkers

Abstract: Background Available blood assays for venous thromboembolism (VTE) suffer from diminished specificity. Compared with single marker tests, such as D-dimer, a multi-marker strategy may improve diagnostic ability. We used direct mass spectrometry (MS) analysis of serum from patients with VTE to determine whether protein expression profiles would predict diagnosis. Methods and Results We developed a direct MS and computational approach to the proteomic analysis of serum. Using this new method, we analyzed serum from inpatients undergoing radiographic evaluation for VTE. In a balanced cohort of 76 patients, a neural network-based prediction model was built using a training subset of the cohort to first identify proteomic patterns of VTE. The proteomic patterns were then validated in a separate group of patients within the cohort. The model yielded a sensitivity of 68% and specificity of 89%, which exceeded the specificity of D-dimer assay tested by latex agglutination, ELISA, and immunoturbimetric methods (sensitivity/specificity of 63.2%/60.5%, 97.4%/21.1%, 97.4%/15.8%, respectively). We validated differences in protein expression between patients with and without VTE using more traditional gel-based analysis of the same serum samples. Conclusion Protein expression analysis of serum using direct MS demonstrates potential diagnostic utility for VTE. This pilot study is the first such direct MS study to be applied to a cardiovascular disease. Differences in protein expression were identified and subsequently validated in a separate group of patients. The findings in this initial cohort can be evaluated in other independent cohorts, including patients with inflammatory conditions and chronic (but not acute) VTE, for the diagnosis of VTE.

Peptide antibody depletion and its application to mass spectrometry sample preparation

Abstract: The present invention relates, eg, to a method for pre-processing a sample for mass spectral analysis, comprising cleaving proteins in the sample to peptides and immunodepleting highly abundant and/or well-ionizing and/or proteotypic peptides from the sample Also described are methods for identifying well-ionizing peptides for use in this and other methods; analytic (diagnostic) methods using antibodies against highly ionizable peptides from a protein target of interest; and compositions, kits and devices comprising antibodies of the invention

Unraveling the Complexity of Circulating Forms of Brain Natriuretic Peptide

Abstract: Determining the circulating form of a biomarker is critical for the development of a highly sensitive and specific immunoassay. The hormone brain natriuretic peptide (BNP) is produced in the atria as a preprohormone, and the cleavage of its signal sequence produces the circulating prohormone proBNP (amino acid residues 1–108, human sequence). The prohormone is subsequently cut to yield a 76-amino acid N-terminal (NT) fragment (NT-proBNP) and the 33-amino acid active hormone BNP (comprising residues 77–108 of proBNP). The 2 commercially available immunoassays detect either the circulating NT fragment or BNP and, potentially, could detect proBNP. However, whether these assays detect all possible circulating forms of BNP remains unclear. The potential circulating forms of BNP have different intrinsic physical characteristics that can dictate antigenicity, extent of nonspecific binding during isolation, endogenous clearance kinetics, circulating half-life, and/or affinity for other proteins including cellular receptors and putative serum/plasma carrier proteins. Consequently, characterization of the physical status of each circulating form of BNP is key to assay development. It is critical to determine whether the circulating BNP forms have posttranslational modifications (PTM), such as phosphorylation or N- and O-linked glycosylation, and/or bind to other protein(s). In a recent issue of Clinical Chemistry , an article by Seferian et al. (1) described 2 approaches to shedding light on the circulating forms of BNP. The 2 strategies examined differences in ( a ) antigenicity, ( b ) antibody binding, and ( c ) the theoretical and observed MWs of the unmodified recombinant form and the circulating endogenous form(s). The data presented indicated that the chemistry of circulating BNP is complex. This complexity needs to be taken into account during immunoassay development. Is there a potential PTM? Seferian et al. (1) produced a large number of peptides and fragments of BNP (pro, NT-pro, and BNP) …

Optimization of Cardiac Troponin I Pull-Down by Idm Affinity Beads and SELDI-TOF

Abstract: Cardiac troponin I (cTnI) is a key regulator of cardiac muscle contraction. Upon myocardial cell injury, cTnI is lost from the cardiac myocyte and can be detected in serum, in some cases with specific disease-induced modifications, making it an important diagnostic marker for acute myocardial injury. Presently, hospital laboratories use enzyme-linked immunosorbent assays to detect cTnI, but this type of analysis lacks information about modified forms of protein (degradation or phosphorylation) that may give a more specific diagnosis from either serum or biopsies. Because cardiac and serum tissues are widely used for proteomic analysis, it is important to detect these cTnI posttranslational modifications. Therefore, we have chosen to optimize the enrichment and detection of cTnI protein by IDM Affinity Bead pull-down and surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS or SELDI) analyses. By adjusting the chemical compositions of the buffers, we have retained antibody specificity and enriched for different forms of cTnI and its associated proteins.

A solubility optimization protocol for two-dimensional gel electrophoresis of cardiac tissue.

Abstract: We outline a strategy for the optimization of buffer conditions for the solubilization, extraction, and isoelectric focusing (IEF) of proteins from cardiac tissue for two-dimensional gel electrophoresis (2DE). This strategy, which involves altering both the extraction and IEF buffers, allows one to ensure representation of the proteome that is as complete as possible. Initial buffer choices are given, as well as basic protocols for modifications. Although these conditions have been effectively demonstrated for human myocardium, in principle this procedure can be used for the initial screen of any new sample of tissue or cultured cells.

A method for the effective depletion of albumin from cellular extracts: application to human myocardium.

Abstract: Proteomic analysis of large numbers of proteins is assisted if each protein species is present at approximately equal concentrations. As such, the extraction of proteins from tissue samples should be designed to maintain a limited dynamic range in the concentration of proteins present. However, in many tissue extracts a high concentration of serum albumin exists from tissue perfusion and/or an inability to effectively rinse the tissue owing to surgical limitations. The analysis of these tissues would be assisted if contaminating serum albumin could be reduced. This chapter outlines a protocol for the effective reduction of serum albumin levels from human myocardium extracts enriched for soluble cytoplasmic proteins.

Abstract 488: Sinoatrial Nodal Pacemaker Cells (SANC) Exhibit High Basal CA Activated Adenylyl Cyclase (AC) Activity and Express Multiple Distinctly Localized AC Types

Abstract: In SANC constituitive AC generates high basal cAMP, inducing PKA-dependent phosphorylation that regulates Ca2+ cycling, that is essential for normal pacemaker function Our goals were to identify,