High-abundance Serum Proteins Research Articles

High-abundance serum glycoproteins as valuable resources for glycopeptide standards

High-abundance serum proteins, mostly modified by N-glycans, are usually depleted from human sera to achieve in-depth analyses of serum proteome and sub-proteomes. In this study, we show that these high-abundance glycoproteins (HAGPs) can be used as valuable standard glycopeptide resources, as long as the structural features of their glycans have been well defined at the glycosite-specific level. By directly analyzing intact glycopeptides enriched from serum, we identified 1322 unique glycopeptides at 48 N-glycosites from the top 12 HAGPs (19 subclasses). These HAGPs could be further classified into four major groups based on the structural features of their attached N-glycans. Immunoglobins including IGHG1/2/3/4, IGHA1/2 and IGHM were mostly modified by core fucosylated and bisected N-glycans with rarely sialic acids. Alpha-1-acid glycoproteins (ORM1/2) and haptoglobins (HP) were mainly modified by tri-and tetra-antennary (40 %) N-glycans with antenna-fucoses and sialic acids. Complement components C3 and C4A/B were highly modified by oligo-mannose glycans. The other HAGPs including SERPINA1, A2M, TF, FGB/G and APOB mainly contain bi-antennary complex glycans with the common core structure and (sialyl-) LacNAc branch structures. These HAGPs are easily detected by LC-MS analysis and therefore could be used as standard glycopeptides for glycoproteomic methodology studies as well as possible clinical utilities.

Enhancing biomarker detection in human serum for lung cancer diagnosis: Aqueous biphasic systems for simultaneous depletion of high-abundance proteins and efficient extraction of CYFRA 21–1

Analysing biomarkers in human serum could be used as an effective and less invasive approach for the diagnosis of lung cancer; however, biomarker detection reliability is highly limited due to matrix effects. Herein, aqueous biphasic systems (ABS) are studied as platforms for human serum pretreatment, allowing the simultaneous depletion of high abundant proteins and biomarker extraction. By using ABS varying the polyethylene glycol (PEG) molecular weight between 400 and 6000 g·mol−1 and adopting phosphate buffer as the other phase-forming component, the depletion of the high abundance serum proteins immunoglobulin G (IgG) and human serum albumin (HSA) is induced at the ABS interphase, through precipitation, forming a three-phase partitioning system (ABS-TPP). Maximum depletion efficiencies of 99 % for IgG and 70 % for HSA were achieved in one step using PEG 1500-based ABS-TPP. On the other hand, lung cancer biomarkers, such as CYFRA 21–1, are extracted to the PEG-rich phase of the same ABS-TPP with recovery yields of 91 %. This work shows that a proper selection of the PEG molecular weight in the ABS composition leads to the efficient depletion of high-abundance proteins and extraction of cancer biomarkers from human serum, in a single step, confirming the potential of ABS for sample pretreatment to improve biomarker analysis.

Improved accuracy in pentraxin-3 quantification assisted by aqueous biphasic systems as serum pretreatment strategies

Although pentraxin-3 holds promise as a diagnosis/prognosis biomarker of microbial infections and lung cancer, its analysis in human serum can be constrained by matrix effects caused by high abundance proteins – human serum albumin and immunoglobulin G. Aqueous biphasic systems composed of polymers and citrate buffer are here proposed as a serum pretreatment step to improve the accuracy of pentraxin-3 analysis. Binodal curves were determined to identify the compositions required to form two phases and to correlate the polymers' properties and performance in serum pretreatment and biomarker extraction. Aqueous biphasic systems were evaluated regarding their ability to deplete human serum albumin and immunoglobulin G at the interphase. Polymers of relatively high to intermediate hydrophobicity were unveiled as efficient components to deplete high abundance serum proteins. Considering the possibility to extract pentraxin-3 from human serum into the polymer-rich phase, the system composed of polyethylene glycol with a molecular weight of 1000 g·mol−1 simultaneously achieved >93 % of human serum albumin and immunoglobulin G depletion and complete biomarker extraction. The accuracy of analysis of pretreated human serum by enzyme-linked immunosorbent assays outperformed that of a non-pretreated sample, with a relative error of 0.8 % compared to 14.6 %, contributing to boost pentraxin-3 usefulness as a biomarker.

Tailored pretreatment of serum samples and biomarker extraction afforded by ionic liquids as constituents of aqueous biphasic systems

Prostate cancer serum biomarkers, such as the U.S. Food and Drug Administration (FDA)-approved prostate specific antigen (PSA), are valuable markers of diagnosis, response to treatment and disease progression. Despite the expected benefits of using PSA to better manage the mortality and morbidity associated to prostate cancer, sample complexity and interference within analysis make its quantification in serum a high-cost and strenuous assignment. Here, we make use of the simple preparation, water-rich environment, and selectivity of aqueous biphasic systems (ABS) to develop alternative serum pretreatment strategies. A set of ammonium- and phosphonium-based ionic liquids (ILs) were used as either main phase-forming agents in IL-salt ABS or as adjuvants in polymer-salt ABS, allowing to explore the IL designer solvent nature and versatile role in ABS formation and performance. Serum pretreatment efficiency was evaluated by the capacity of each ABS to simultaneously deplete high abundance serum proteins (immunoglobulin G, IgG, and human serum albumin, HSA) at the systems’ interphase (thus, generating a three-phase partitioning system – ABS-TPP) and extract PSA to a single ABS phase. Regardless of the ABS typology under appraisal (IL-salt or polymer-salt-IL), the proper design of the IL structure is critical for both the ability to deplete high abundance serum proteins and extract PSA. The results demonstrate that, in addition to the proper choice of the ABS typology, a careful adjustment of the IL hydrophobicity is key to achieve the simultaneous depletion of high-abundance proteins and complete PSA extraction in a single step, creating new approaches for PSA analysis and its application in diagnosis/prognosis.

Tracking Heme-Protein Interactions in Healthy and Pathological Human Serum in Native Conditions by Miniaturized FFF-Multidetection

The interaction of heme with blood serum proteins plays an important role in many physiological and pathological processes involving enzyme activity, gene expression and cell proliferation. The mechanisms underlying these interactions are; however, not yet fully understood. New analytical methods able to investigate protein-heme binding in native, biologically representative conditions are thus required. In this work, we present a method based on miniaturized, hollow-fiber flow field-flow fractionation with multiple spectrophotometric and light-scattering detection for size separation of high-abundance serum proteins and selective detection of heme-bound subpopulations. Heme is found to mainly interact with serum albumin, whereas a low amount also binds to other proteins such as IgM. The ability to bind heme in physiological conditions is also investigated for individual serum proteins. IgG is found unable to bind heme at clinically relevant concentrations. The proposed method allows separation, quantitation, and mass/size characterization of serum high-abundance proteins, providing information of heme-protein complex stability and preferred heme-clearing pathways. The same approach could be in perspective extended to the investigation of specific heme-antibody binding, and to further studies involving other molecules of pharmaceutical/clinical interest.

High Resolution Continuous Elution Electrophoresis for the Evaluation of Low Abundance Serum Proteins.

The evaluation of low abundance biomarkers in the circulating low molecular weight serum proteome is an important source of information. Techniques for sample preparation to remove high abundant proteins and to enrich the low molecular weight fraction are usually required prior to novel biomarker detection. A continuous elution electrophoresis was used to separate the low molecular weight serum proteins from the high abundance serum proteins, such as albumin and immunoglobulins. Centrifugal concentration, SDS-PAGE, and total protein staining were performed to analyze eluted protein fractions. Consecutive concentrated serum protein fractions demonstrate separation at a high resolution of 1 - 2 kDa below 20 kDa. Continuous elution electrophoresis is an adequate method to eliminate high abundance proteins which interfere with the detection of low abundance biomarkers in the low molecular weight proteome and to enrich its proteins for subsequent detection and clinical evaluation.

Associations between the thyroid panel and serum protein concentrations across pregnancy

Establishing any characteristic associations between the serum parameters of thyroid function and serum proteins in pregnancy may aid in elucidating the role of the thyroid gland in the regulation of pregnancy-specific metabolic processes and in selecting candidate biomarkers for use in their clinical assessment. Concentrations of thyroid stimulating hormone (TSH), free tri-iodothyronine (fT3) and free thyroxine (fT4), six electrophoretically separated protein fractions (albumin, alpha-1-, alpha2-, beta-1-, beta-2- and gamma-globulins), representative proteins—albumin (ALB), transferrin (TRF), alpha-2-macroglobulin (AMG) and ceruloplasmin (CER) were measured in 136 serum samples from 65 women in their consecutive trimesters of pregnancy. The concentrations of TSH, fT4 and fT3 were significantly correlated (p < 0.05) with the concentrations of the albumin, alpha-2- and beta-1 globulin fractions. Significant correlations (p < 0.05) which were positive between fT4 and ALB and negative between fT4 and TRF were established throughout pregnancy. Significant negative correlations (p < 0.05) were demonstrated for fT3 with alpha-2-globulin, AMG and CER. Changes in the serum concentrations of thyroid hormones seen between the trimesters were found to correlate with the concentrations of high-abundance serum proteins. Opposite directions of correlations between fT4 and ALB and fT4 and TRF observed throughout pregnancy may indicate the shared biological role of these parameters in maintaining maternal homeostasis and they suggest their potential use in the clinic as a simple biomarker panel. A negative correlation of fT3 with CER in the second trimester possibly reflects their involvement in the active regulation of metabolic processes.

Enhancing Comprehensive Analysis of Secreted Glycoproteins from Cultured Cells without Serum Starvation.

Glycoproteins secreted by cells play essential roles in the regulation of extracellular activities. Secreted glycoproteins are often reflective of cellular status, and thus glycoproteins from easily accessible bodily fluids can serve as excellent biomarkers for disease detection. Cultured cells have been extensively employed as models in the research fields of biology and biomedicine, and global analysis of glycoproteins secreted from these cells provides insights into cellular activities and glycoprotein functions. However, comprehensive identification and quantification of secreted glycoproteins is a daunting task because of their low abundances compared with the high-abundance serum proteins required for cell growth and proliferation. Several studies employed serum-free media to analyze secreted proteins, but it has been shown that serum starvation, even for a short period of time, can alter protein secretion. To overcome these issues, we developed a method to globally characterize secreted glycoproteins and their N-glycosylation sites from cultured cells by combining selective enrichment of secreted glycoproteins with a boosting approach. The results demonstrated the importance of the boosting sample selection and the boosting-to-sample ratio for improving the coverage of secreted glycoproteins. The method was applied to globally quantify secreted glycoproteins from THP-1 monocytes and macrophages in response to lipopolysaccharides (LPS) and from Hep G2 cells treated with TGF-β without serum starvation. We found differentially secreted glycoproteins in these model systems that showed the cellular response to the immune activation or the epithelial-to-mesenchymal transition. Benefiting from the selective enrichment and the signal enhancement of low-abundance secreted glycoproteins, this method can be extensively applied to study secreted glycoproteins without serum starvation, which will provide a better understanding of protein secretion and cellular activity.

Proteome Profiling of Exosomes Purified from a Small Amount of Human Serum: The Problem of Co-Purified Serum Components.

Untargeted proteomics analysis of extracellular vesicles (EVs) isolated from human serum or plasma remains a technical challenge due to the contamination of these vesicles with lipoproteins and other abundant serum components. Here we aimed to test a simple method of EV isolation from a small amount of human serum (<1 mL) using the size-exclusion chromatography (SEC) standalone for the discovery of vesicle-specific proteins by the untargeted LC–MS/MS shotgun approach. We selected the SEC fraction containing vesicles with the size of about 100 nm and enriched with exosome markers CD63 and CD81 (but not CD9 and TSG101) and analyzed it in a parallel to the subsequent SEC fraction enriched in the lipoprotein vesicles. In general, there were 267 proteins identified by LC–MS/MS in exosome-containing fraction (after exclusion of immunoglobulins), yet 94 of them might be considered as serum proteins. Hence, 173 exosome-related proteins were analyzed, including 92 proteins absent in lipoprotein-enriched fraction. In this set of exosome-related proteins, there were 45 species associated with the GO cellular compartment term “extracellular exosome”. Moreover, there were 31 proteins associated with different immune-related functions in this set, which putatively reflected the major role of exosomes released by immune cells present in the blood. We concluded that identified set of proteins included a bona fide exosomes components, yet the coverage of exosome proteome was low due to co-purified high abundant serum proteins. Nevertheless, the approach proposed in current work outperformed other comparable protocols regarding untargeted identification of exosome proteins and could be recommended for pilot exploratory studies when a small amount of a serum/plasma specimen is available.

Harmonization of exosome isolation from culture supernatants for optimized proteomics analysis.

Exosomes, the smallest subset of extracellular vesicles (EVs), have recently attracted much attention in the scientific community. Their involvement in intercellular communication and molecular reprogramming of different cell types created a demand for a stringent characterization of the proteome which exosomes carry and deliver to recipient cells. Mass spectrometry (MS) has been extensively used for exosome protein profiling. Unfortunately, no standards have been established for exosome isolation and their preparation for MS, leading to accumulation of artefactual data. These include the presence of high-abundance exosome-contaminating serum proteins in culture media which mask low-abundance exosome-specific components, isolation methods that fail to yield “pure” vesicles or variability in protein solubilization protocols. There is an unmet need for the development of standards for exosome generation, harvesting, and isolation from cellular supernatants and for optimization of protein extraction methods before proteomics analysis by MS. In this communication, we illustrate the existing problems in this field and provide a set of recommendations that are expected to harmonize exosome processing for MS and provide the faithful picture of the proteomes carried by exosomes. The recommended workflow for effective and specific identification of proteins in exosomes released by the low number of cells involves culturing cells in medium with a reduced concentration of exosome-depleted serum, purification of exosomes by size-exclusion chromatography, a combination of different protein extraction method and removal of serum-derived proteins from the final dataset using an appropriate sample of cell-unexposed medium as a control. Application of this method allowed detection of >250 vesicle-specific proteins in exosomes from 10 mL of culture medium.

Analytical validation of protein biomarkers for risk of spontaneous preterm birth

Presented are the validation results of a second-generation assay for determining the relative abundances of two protein biomarkers found in maternal serum that predict an individual’s risk of spontaneous preterm birth. The sample preparation workflow is complex, consisting of immuno-depletion of high-abundance serum proteins, tryptic digestion of the immuno-depleted fraction to generate surrogate peptide analytes, and detection by tandem mass spectrometry. The method was determined to be robust on observation of the following characteristics: classifier peptide detection precision was excellent; results were accurate when compared to a reference method; results were linear over a clinically relevant range; the limits of quantitation encompassed the range of expected results; and the method demonstrated analytical specificity and resilience to differences in patient serum and common endogenous interferents.

The use of Nanotrap particles in the enhanced detection of Rift Valley fever virus nucleoprotein.

BackgroundRift Valley fever virus (RVFV) is a highly pathogenic arthropod-borne virus that has a detrimental effect on both livestock and human populations. While there are several diagnostic methodologies available for RVFV detection, many are not sensitive enough to diagnose early infections. Furthermore, detection may be hindered by high abundant proteins such as albumin. Previous findings have shown that Nanotrap particles can be used to significantly enhance detection of various small analytes of low abundance. We have expanded upon this repertoire to show that this simple and efficient sample preparation technology can drastically improve the detection of the RVFV nucleoprotein (NP), the most abundant and widely used viral protein for RVFV diagnostics.ResultsAfter screening multiple Nanotrap particle architectures, we found that one particle, NT45, was optimal for RVFV NP capture, as demonstrated by western blotting. NT45 significantly enhanced detection of the NP at levels undetectable without the technology. Importantly, we demonstrated that Nanotrap particles are capable of concentrating NP in a number of matrices, including infected cell lysates, viral supernatants, and animal sera. Specifically, NT45 enhanced detection of NP at various viral titers, multiplicity of infections, and time points. Our most dramatic results were observed in spiked serum samples, where high abundance serum proteins hindered detection of NP without Nanotrap particles. Nanotrap particles allowed for sample cleanup and subsequent detection of RVFV NP. Finally, we demonstrated that incubation of our samples with Nanotrap particles protects the NP from degradation over extended periods of time (up to 120 hours) and at elevated temperatures (at 37ºC).ConclusionThis study demonstrates that Nanotrap particles are capable of drastically lowering the limit of detection for RVFV NP by capturing, concentrating, and preserving RVFV NP in clinically relevant matrices. These studies can be extended to a wide range of pathogens and their analytes of diagnostic interest.

Absolute quantification of DcR3 and GDF15 from human serum by LC-ESI MS.

Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high-abundant serum proteins by partial denaturation and enrichment of low-abundant biomarkers by size exclusion chromatography. The recovery of low-abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 μl human serum by isotope dilution mass spectrometry, using 15N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody-based strategies, and offers the possibility of multiplexing. Our proof-of-principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts.

Binding between lead ions and the high-abundance serum proteins

The interaction between three of the most abundant bovine serum proteins (serum albumin, transferrin and IgG) with Pb2+ was investigated using electrochemistry. The data was used to construct a new theoretical model of Pb2+ binding to the high-abundance serum proteins under non-ideal conditions. The binding constants (β) of Pb2+ to the individual proteins and a mixture of proteins were measured according to a new theoretical equation (non-ideal state) as well as the McGhee–Von Hippel equation (ideal state). Differences between the models suggested that the β values obtained using the non-ideal state model was more realistic. Protein–protein interactions and micro-environmental influences affected binding between Pb2+ and the high-abundance serum proteins. We included a micro-environmental influence factor for the model (Fm), which accurately quantified the effect of micro-environment of the proteome of Pb2+ binding with the serum proteins. This research provides a useful reference of theoretical and experimental work regarding heavy-metal binding interactions with serum proteins.

Development of a highly sensitive liquid chromatography/tandem mass spectrometry method to quantify total and free levels of a target protein, interferon-gamma-inducible protein-10, at picomolar levels in human serum.

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays are increasingly being used for absolute quantitation of proteins due to high specificity and low cost. However, the major challenge for the LC/MS method is insufficient sensitivity. This paper details the strategies developed to maximize the sensitivity from aspects of chromatography, mass spectrometry, and sample preparation to achieve a highly sensitive LC/MS method. The method is based on the LC/MS/MS measurement of a surrogate peptide generated from trypsin digestion of interferon-gamma-inducible protein-10 (IP-10). The sample preparation strategy involved selectively extracting IP-10 and removing high-abundance serum proteins through acidified protein precipitation (PPT). It was revealed in this work that these high-abundance serum proteins, if not separated from the protein of interest, could cause significant ionization saturation and high background noise in selected reaction monitoring (SRM), leading to a 100-fold higher lower limit of quantification (LLOQ). Our method demonstrated that the acidified PPT could be optimized to selectively extract the protein of interest with full recovery of 97% to 103%, while the high-abundance serum proteins could be effectively removed with minimal matrix effect of 90% to 93%. For the first time, a highly sensitive LC/MS method with a LLOQ of 31.62 pM for the quantitation of IP-10 has been achieved, which is a 100-fold improvement over the generic method. The described method offers excellent sensitivity with advantages of being antibody reagent independent and leads to significant cost and time savings. It has been successfully employed to determine both total and free IP-10 levels in human serum samples. This method development strategy may also be applied to other small proteins.

Comparative proteomic study in serum of patients with primary open-angle glaucoma and pseudoexfoliation glaucoma

Alterations in the sera proteins between patients with Primary Open-Angle Glaucoma (POAG), Pseudoexfoliation Glaucoma (PEXG), and healthy controls were identified through a proven approach utilizing equalization of high-abundance serum proteins with ProteoMiner™, two-dimensional fluorescent difference gel electrophoresis (2D-DIGE), MALDI-TOF/TOF, and nanoLC-MS-MS. Quantitative immunoassays of the 17 most-differentially-altered proteins identified in this analysis confirmed that they were also over expressed in the intact serum of newly recruited glaucoma patients. Overall, this report identifies a panel of candidates for glaucoma biomarkers and supports their further validation in large population studies. Additionally, functional pathway analysis of these candidate proteins suggested that they are part of a network linked to regulating immune and inflammatory-related processes. The data have been deposited to the ProteomeXchange with identifier PXD000198. POAG and PEXG are major causes of age-related blindness in the world; however, treatment can be very effective if they are identified early on in the progression. Genetic linkage studies can only explain a limited number of cases, suggesting that these forms of glaucoma are multigenic in nature. Other important factors, such as modifier genes, epigenetic influences, environmental and dietary agents, and inflammatory and oxidative effects are also believed to affect the development of these diseases. The characterization of metabolic and/or proteins changes, for example in bodily fluids, before the clinical manifestation of glaucoma is of considerable relevance for its early diagnosis. In the present work, identification of over-expressed proteins in serum of glaucoma patients (POAG and PEXG) linked to immune and inflammatory processes supports the finding that changes in these pathways also manifest systemically in patients with these pathologies. This study provides a new basis to validate the identified proteins as biomarkers of glaucoma in a large-scale-multiplexed screening in sera.

Development of candidate biomarkers for pancreatic ductal adenocarcinoma using multiple reaction monitoring

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most frequent cause of cancer mortality in the United States. Because CA 19-9 increases not only in PDAC, but also in benign conditions, there is urgent need for an additional PDAC biomarker. Isotope tags for relative and absolute quantification (iTRAQ) were performed using 6 pairs of PDAC and normal tissues from the same patients, to obtain preliminary PDAC-specific proteins; and verification was performed by multiple reactions monitoring (MRM), using 30 PDAC and 20 normal serum, targeting high-abundant serum proteins without any pre-preparation. As a result, 17 candidate proteins from tissue iTRAQ were verified as potential markers (AUC values > 0.7). Multivariate analysis (MA) demonstrated that a 6-marker panel, consisting of alpha-1 antitrypsin, haptoglobin beta chain, hemopexin, transferrin, zinc alpha-2 glycoprotein, and apolipoprotein A4 from the MRM result, had comparable discriminatory power versus CA 19-9. Our study demonstrated that a combination of iTRAQ on PDAC tissue and verification MRM-MA on individual serum was an efficient method for the development of PDAC multimarkers.

Serum Proteomic Signature of Human Chagasic Patients for the Identification of Novel Potential Protein Biomarkers of Disease

Chagas disease is initiated upon infection by Trypanosoma cruzi. Among the health consequences is a decline in heart function, and the pathophysiological mechanisms underlying this manifestation are not well understood. To explore the possible mechanisms, we employed IgY LC10 affinity chromatography in conjunction with ProteomeLab PF2D and two-dimensional gel electrophoresis to resolve the proteome signature of high and low abundance serum proteins in chagasic patients. MALDI-TOF MS/MS analysis yielded 80 and 14 differentially expressed proteins associated with cardiomyopathy of chagasic and other etiologies, respectively. The extent of oxidative stress-induced carbonyl modifications of the differentially expressed proteins (n = 26) was increased and coupled with a depression of antioxidant proteins. Functional annotation of the top networks developed by ingenuity pathway analysis of proteome database identified dysregulation of inflammation/acute phase response signaling and lipid metabolism relevant to production of prostaglandins and arachidonic acid in chagasic patients. Overlay of the major networks identified prothrombin and plasminogen at a nodal position with connectivity to proteome signature indicative of heart disease (i.e., thrombosis, angiogenesis, vasodilatation of blood vessels or the aorta, and increased permeability of blood vessel and endothelial tubes), and inflammatory responses (e.g., platelet aggregation, complement activation, and phagocyte activation and migration). The detection of cardiac proteins (myosin light chain 2 and myosin heavy chain 11) and increased levels of vinculin and plasminogen provided a comprehensive set of biomarkers of cardiac muscle injury and development of clinical Chagas disease in human patients. These results provide an impetus for biomarker validation in large cohorts of clinically characterized chagasic patients.

Investigating the secretome: lessons about the cells that comprise the heart.

The cell/environment interface is composed of the proteins of plasma membrane which face the extracellular space and by the proteins secreted directly by the cell of origin or by neighboring cells. The secreted proteins can act as extracellular matrix proteins and/or autocrine/paracrine proteins. This report discusses the technical aspects involved in the identification and characterization of the secreted proteins of specific cell types that comprise the heart. These aspects include the culturing of the cells, cell co-culturing and quantitative labeling, conditioned media collection and dealing with high abundant serum proteins, post-translational modification enrichment, the use of protein separation methods and mass spectrometry, protein identification and validation and the incorporation of pathway analysis to better understand the novel discovery on the background of already known experimental biological systems. The proteomic methods have the solid emplacement in cardiovascular research and the identification of proteins secreted by cardiac cells has been used in various applications such as determination the specificity between secretomes of different cell types, e.g. cardiac stem cells and cardiac myocytes, for the global secretome screening of e.g. human arterial smooth muscle cells, for the mapping of the beneficial effect of conditioned medium of one cell type on the other cell type, e.g. conditioned medium of human mesenchymal stem cells on cardiac myocytes, and for the searching the candidate paracrine factors and potential biomarkers.

Hydrophilic diol monolith for the preparation of immuno‐sorbents at reduced nonspecific interactions

A polar organic polymer monolith (M1) was introduced for performing immunoaffinity chromatography (IAC) at reduced nonspecific interactions. The M1 monolith was prepared by the in situ polymerization of glyceryl methacrylate (GMM) and pentaerythritol triacrylate (PETA). Through its surface diol groups, M1 provided the functionalities to immobilize antibodies. Anti-haptoglobin antibody was used as the model antibody to study the overall behavior of the immuno monolith M1 in terms of its binding to the antigen and to evaluate its nonspecific binding with other proteins, especially the high-abundance human serum proteins. To better assess the suitability of M1 for IAC, other immuno monoliths were prepared and compared with the immuno monolith M1. Two monoliths were of the traditional ones: copolymers of (i) glycidyl methacrylate and ethylene glycol dimethacrylate (EDMA) and (ii) GMM and EDMA, referred to as M2 and M3, respectively. A fourth monolith involving the copolymerization of N-(3-aminopropyl)methacrylamide hydrochloride and EDMA (M4) was introduced to allow the site-directed immobilization of antibodies. Owing to its hydroxyl groups, the M1 exhibited negligible nonspecific hydrophobic interactions with proteins. On the other hand, M4 exhibited extensive electrostatic interactions, while the M2 and to a lesser extent M3 exhibited hydrophobic interactions.