Search

Background The anatomy of PFO suggests that it can allow thrombi and potentially harmful circulatory factors to travel directly from the venous to the arterial circulation - altering circulatory phenotype. Our previous publication using high-resolution LC-MS/MS to profile protein and peptide expression patterns in plasma showed that albumin was relatively increased in donor samples from PFO-related than other types of ischemic strokes. Since albumin binds a host of molecules and acts as a carrier for lipoproteins, small molecules and drugs, we decided to investigate the albumin-bound proteins (in a similar sample cohort) in an effort to unravel biological changes and potentially discover biomarkers related to PFO-related stroke and PFO endovascular closure. Methods The method used in this study combined albumin immuno-enrichment with high resolution LC-MS in order to specifically capture and quantify the albumin-bound proteins. Subsequently, we measured cholesterol and HDL in a larger, separate cohort of PFO stroke patients, pre and post closure. Results The results demonstrated that a number of proteins were specifically associated with albumin in samples with and without endovascular closure of the PFO, and that the protein profiles were very different. Eight proteins, typically associated with HDL were common to both sample sets and quantitatively differently abundant. Pathway analysis of the MS results suggested that enhanced cholesterol efflux and reduced lipid oxidation were associated with PFO closure. Measurement of total cholesterol and HDL in a larger cohort of PFO closure samples using a colorimetric assay was consistent with the proteomic predictions. Conclusions The collective data presented in this study demonstrate that analysis of albumin-bound proteins could provide a valuable tool for biomarker discovery on the effects of PFO endovascular closure. In addition, the results suggest that PFO endovascular closure can potentially have effects on HDL, cholesterol and albumin-bound ApoA-I abundance, therefore possibly providing benefits in cardioprotective functions. [ABSTRACT FROM AUTHOR]

Patent foramen ovale (PFO) is highly prevalent and associated with more than 150,000 strokes per year. Traditionally, it is thought that PFOs facilitate strokes by allowing venous clots to travel directly to the brain. However, only a small portion of PFO stroke patients have a known tendency to form blood clots, and the optimal treatment for this multiorgan disease is unclear. Therefore, mapping the changes in systemic circulation of PFO-related stroke is crucial in understanding the pathophysiology to individualize the best clinical treatment for each patient. We initiated a study using a novel quantitative, 2-pass discovery workflow using high-resolution liquid chromatography-mass spectrometry/mass spectrometry coupled with label-free analysis to track protein expression in PFO patients before and after endovascular closure of the PFO. Using this approach, we were able to demonstrate quantitative differences in protein expression between both PFO-related and non-PFO-related ischemic stroke groups as well as before and after PFO closure. As an initial step in understanding the molecular landscape of PFO-related physiology, our methods have yielded biologically relevant information on the synergistic and functional redundancy of various cell-signaling molecules with respect to PFO circulatory physiology. The resulting protein expression patterns were related to canonical pathways including prothrombin activation, atherosclerosis signaling, acute-phase response, LXR/RXR activation, and coagulation system.In particular, after PFO closure, numerous proteins demonstrated reduced expression in stroke-related canonical pathways such as acute inflammatory response and coagulation signaling. These findings demonstrate the feasibility and robustness of using a proteomic approach for biomarker discovery to help gauge therapeutic efficacy in stroke. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. Design and methods: The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. Results: In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer''s, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67–0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. Conclusions: We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications. [Copyright &y& Elsevier]

Abstract: Because accumulation of α-synuclein (αS) in the brain is a hallmark of Parkinson disease (PD) and related disorders, we examined its occurrence in human cerebrospinal fluid (CSF). Following affinity enrichment and trypsin digestion of CSF collected from a neurologically healthy donor, we identified several αS-derived peptides by mass spectrometry. The concentration of αS amounted to <0.001% of the CSF proteome. We then built, validated and optimized a sandwich-type, enzyme-linked immunoadsorbent assay (ELISA) to measure total αS levels in unconcentrated CSF. In a cross-sectional study of 100 living donors, we examined cell-free CSF samples from subjects clinically diagnosed with advanced PD, dementia with Lewy bodies (DLB), Alzheimer disease (AD), and a group of non-neurodegenerative disease controls (NCO). In these four groups the CSF αS concentrations ranged from 0.8 to 16.2 pg/μl. Mean CSF αS values were lower in donors with a primary synucleinopathy (PD, DLB: n =57) than in the other two groups (AD, NCO: n =35; p =0.025). By contrast, living Creutzfeldt–Jakob disease patients showed markedly elevated CSF αS levels (n =8; mean, 300 pg/μl; p <0.001). Our results unequivocally confirm the presence of αS in adult human CSF. In a first feasibility study employing a novel ELISA, we found relatively low CSF αS concentrations in subjects with parkinsonism linked to synucleinopathy, PD and DLB. In definite prion disease cases, we recorded a marked rise in total CSF αS resulting from rapid cell death. Our results will likely aid future biomarker explorations in neurodegenerative conditions and facilitate target validation studies. [Copyright &y& Elsevier]