Proteomic analysis of plasma-purified VLDL, LDL, and HDL fractions from atherosclerotic patients undergoing carotid endarterectomy: identification of serum amyloid A as a potential marker.

Apolipoproteins are very heterogeneous protein family, implicated in plasma lipoprotein structural stabilization, lipid metabolism, inflammation, or immunity. Obtaining detailed information on apolipoprotein composition and structure may contribute to elucidating lipoprotein roles in atherogenesis and to developing new therapeutic strategies for the treatment of lipoprotein-associated disorders. This study aimed at developing a comprehensive method for characterizing the apolipoprotein component of plasma VLDL, LDL, and HDL fractions from patients undergoing carotid endarterectomy, by means of two-dimensional electrophoresis (2-DE) coupled with Mass Spectrometry analysis, useful for identifying potential markers of plaque presence and vulnerability. The adopted method allowed obtaining reproducible 2-DE maps of exchangeable apolipoproteins from VLDL, LDL, and HDL. Twenty-three protein isoforms were identified by peptide mass fingerprinting analysis. Differential proteomic analysis allowed for identifying increased levels of acute-phase serum amyloid A protein (AP SAA) in all lipoprotein fractions, especially in LDL from atherosclerotic patients. Results have been confirmed by western blotting analysis on each lipoprotein fraction using apo AI levels for data normalization. The higher levels of AP SAA found in patients suggest a role of LDL as AP SAA carrier into the subendothelial space of artery wall, where AP SAA accumulates and may exert noxious effects.

Analysis of changes in tyrosine and serine phosphorylation of red cell membrane proteins induced by P. falciparum growth.

Phosphorylation of erythrocyte membrane proteins has been previously documented following infection and intracellular growth of the malarial parasite, Plasmodium falciparum in red cells. Much of this data dealt with phosphorylation of serine residues. In this study, we report detailed characterization of phosphorylation of serine and tyrosine residues of red cell membrane proteins following infection by P falciparum. Western blot analysis using anti-phosphotyrosine and anti-phosphoserine antibodies following 2-DE in conjunction with double channel laser-induced infrared fluorescence enabled accurate assessment of phosphorylation changes. Tyrosine phosphorylation of band 3 represented the earliest modification observed during parasite development. Band 3 tyrosine phosphorylation observed at the ring stage appears to be under the control of Syk kinase. Serine and tyrosine phosphorylation of additional cytoskeletal, trans-membrane and membrane associated proteins was documented as intracellular development of parasite progressed. Importantly, during late schizont stage of parasite maturation, we observed widespread protein dephosphorylation. In vitro treatments that caused distinct activation of red cell tyrosine and serine kinases elicited phosphorylative patterns similar to what observed in parasitized red blood cell, suggesting primary involvement of erythrocyte kinases. Identification of tyrosine phosphorylations of band 3, band 4.2, catalase and actin which have not been previously described in P. falciparum infected red cells suggests new potential regulatory mechanisms that could modify the functions of the host cell membrane.

Differential distribution of structural components and hydration in aortic and pulmonary heart valve conduits: Impact of detergent-based cell removal.

Evaluation of the physiological performance of biological scaffolds for tissue engineering applications has been mostly based on biophysical and morphological methods, with limited attention paid to the quantitative contribution of the main structural components to native and/or treated valve assemblies. In the present study quantitation addressed the porcine leaflet, sinus and adjacent wall of aortic and pulmonary valved conduits before and after detergent-based cell removal. Collagen, elastin, glycosaminoglycan, lipid and water contents were expressed in terms of relative concentration and volume fraction in order to assess their effective contribution to the native tissue and to changes following decellularization procedures. The main findings were recognition of unexpectedly large water and underestimated collagen contents, differential distribution of elastin between the sectors and of glycosaminoglycan along the conduits and pulmonary scaffold destabilization upon cell removal, not found in the aortic case. Simultaneous investigations allowed consistent comparisons between native and decellularized tissues and added analytical knowledge crucial for designing realistic constitutive models. We have provided a quantitative structural foundation for earlier biomechanical findings in pulmonary leaflets and the basis for validation of theoretical assumptions still lacking the support of experimental evidence in both conduits. Future insights into the distribution of load-bearing components in human conduits are likely to provide indications important to optimize the surgical positioning of valvular grafts.

A proteomic approach to differentiate histologically classified stable and unstable plaques from human carotid arteries.

OBJECTIVES: By using proteomics we isolated and identified proteins that were expressed/retained in stable and unstable human carotid artery atherosclerotic plaques. METHODS: The criteria for plaque instability were the presence of a thin fibrous cap or fissured cap covering the foamy or necrotic core, and the presence of overt, hemorrhagic, ulcerated or thrombotic plaques. Proteins were extracted from finely minced endarterectomy specimens (19 stable and 29 unstable plaques) and separated by two-dimensional gel electrophoresis. Coomassie Blue-stained gels were analysed using PD-Quest software. RESULTS: A total of 57 distinct spots corresponding to 33 different proteins were identified by matrix assisted laser desorption/ionization mass spectrometry using the NCBI database. Most of the spots were present in both types of extracts, although significantly (p<0.05) differing in abundance between them. Compared to stable plaque, unstable ones showed reduced abundance of: protective enzymes SOD3 and GST, small heat shock proteins HSP27 and HSP20, annexin A10, and Rho GDI. In unstable plaques the more abundant proteins were: ferritin light subunit, SOD 2 and fibrinogen fragment D. For fibrinogen fragment D, the increased levels in unstable versus stable plaques was confirmed by Western blot analysis. CONCLUSIONS: Since many of the differentially expressed proteins are known to have a functional role in inflammation and oxidative stress, we speculate that they may be involved in events relating to plaque stability.

Short-end injection capillary electrophoresis for quantification of plasma chondroitin sulfate isomer disaccharides.

We describe a new ultra-rapid capillary electrophoresis method with UV detection for analysis of the disaccharides obtained after enzymatic depolymerization of plasma chondroitin sulfates. The free reducing groups of the released carbohydrate molecules are derivatized with 2-aminoacridone by reductive amination in the presence of cyanoborohydride. The fluorotagged products can be separated by short-end injection capillary electrophoresis in a capillary with an effective length of 10.2 cm. The migration times of Delta di-0S and Delta di-4S were 0.95 and 1.81 min, respectively. We compared the proposed method with UV detection to a reference CE-LIF assay by measuring plasma chondroitin sulfate in 94 subjects. The described assay for total plasma CS measurement may, owing to the high throughput and the fast analytical times, be a good tool for routine studies both in research and in clinical applications.

Applications of Two-Dimensional Electrophoresis Technology to the Study of Atherosclerosis.

Atherosclerosis is a multifactorial disease in which hypertension, diabetes, hyperlipidemia and other risk factors are thought to play a role. However, the molecular processes underlying plaque formation and progression are not yet completely known. In the last years some researchers applied proteomics technologies for the comprehension of biochemical pathways of atherogenesis and to search new cardiovascular biomarkers to be utilized either as early diagnostic traits or as targets for new drug therapies. Due to its intrinsic complexity, the problem has been approached by different strategies, all of which have some limitations. In this review, we summarize the most common critical experimental variables in two-dimensional electrophoresis-based techniques and recent data obtained by applying proteomic approaches in the study of atherosclerosis.

A novel LIF-CE method for the separation of hyaluronan- and chondroitin sulfate-derived disaccharides: Application to structural and quantitative analyses of human plasma low- and high-charged chondroitin sulfate isomers.

The report describes a rapid and simple CE method using LIF detection for the analysis of unsaturated disaccharides obtained from enzymatic depolymerization of plasma chondroitin sulfate (CS) isomers. The disaccharide reducing groups were labeled with 2-aminoacridone (AMAC). The fluorotagged products can be separated by reversed-polarity CE using a sodium acetate buffer, pH 3.8, in the presence of 0.05% methylcellulose. The choice of the appropriate electrophoretic conditions was performed after a deep analysis of the most important parameters affecting analyte separation. In particular, the effect of both run buffer concentration and pH on resolution, efficiency, migration times, and peak area was evaluated. The selected electrophoretic conditions allowed us to separate the CS isomers-derived Delta-disaccharides in less than 12 min, also resolving the nonsulfated disaccharides released from CS isomers from those released from hyaluronan (HA). Moreover, these conditions gave a good reproducibility of both the migration times (CV%, 0.25) and the peak areas (CV%, 1.4). Intra- and interassay CV were 5.37 and 7.23%, respectively, and analytical recovery was about 86%. The applicability of the above method to the quantitative and structural disaccharide analyses of plasma CS isomers was investigated. Data obtained from 44 healthy human subjects were compared with those obtained by a fluorophore-assisted carbohydrate electrophoresis (FACE) reference assay, by using the Passing and Bablok regression and Bland-Altman tests. The developed method could represent a good tool for an ultrasensitive analysis of CS isomers in biological samples from different sources, particularly when samples are available in very low amounts.