Miroslava Stastna

Secreted proteins as a fundamental source for biomarker discovery

The proteins secreted by various cells (the secretomes) are a potential rich source of biomarkers as they reflect various states of the cells at real time and at given conditions. To have accessible, sufficient and reliable protein markers is desirable as they mark various stages of disease development and their presence/absence can be used for diagnosis, prognosis, risk stratification and therapeutic monitoring. As direct analysis of blood/plasma, a common and noninvasive patient screening method, can be difficult for candidate protein biomarker identification, the alternative/complementary approaches are required, one of them is the analysis of secretomes in cell conditioned media in vitro. As the proteins secreted by cells as a response to various stimuli are most likely secreted into blood/plasma, the identification and pre‐selection of candidate protein biomarkers from cell secretomes with subsequent validation of their presence at higher levels in serum/plasma is a promising approach. In this review, we discuss the proteins secreted by three progenitor cell types (smooth muscle, endothelial and cardiac progenitor cells) and two adult cell types (neonatal rat ventrical myocytes and smooth muscle cells) which can be relevant to cardiovascular research and which have been recently published in the literature. We found, at least for secretome studies included in this review, that secretomes of progenitor and adult cells overlap by 48% but the secretomes are very distinct among progenitor cell themselves as well as between adult cells. In addition, we compared secreted proteins to protein identifications listed in the Human Plasma PeptideAtlas and in two reports with cardiovascular‐related proteins and we performed the extensive literature search to find if any of these secreted proteins were identified in a biomarker study. As expected, many proteins have been identified as biomarkers in cancer but 18 proteins (out of 62) have been tested as biomarkers in cardiovascular diseases as well.

Identification and functionality of proteomes secreted by rat cardiac stem cells and neonatal cardiomyocytes

In the heart, the proteomes secreted by both cardiac stem cells (CSCs) and cardiac myocytes could act synergistically, but the identification and functionality of the proteins comprising the individual secretomes have not yet been described. In this study, we have identified proteins present in the media obtained from cultured rat CSCs and from cultured neonatal rat ventricular myocytes (NRVMs) and compared them with proteins identified in the media alone. Briefly, 83 unique proteins were identified after analysis by RPLC and MS. In total 49 and 23% were NRVM‐specific or CSC‐specific proteins, respectively, and 63% of total 83 proteins were integral plasma membrane and/or known secreted proteins. Fifteen proteins met our criteria for paracrine/autocrine factors: (i) robust protein identification, (ii) cell specific and (iii) known to be secreted. Most of these proteins have not been previously linked to stem cells. NRVM‐specific proteins atrial natriuretic factor (ANP) and connective tissue growth factor, and CSC‐specific protein interleukin‐1 receptor‐like 1 (ST2) were found to affect rat CSC proliferation. These findings suggest that relative concentration of each protein may be crucial for cellular intertalk and for the final outcome of cardiac cell therapy.

Cardiac stem/progenitor cells, secreted proteins, and proteomics

Stem cell‐based therapy is emerging as a novel approach for myocardial repair over conventional cardiovascular therapies. In addition to embryonic stem cells and adult stem cells from noncardiac sources, there is a small population of resident stem cells in the heart from which new cardiac cells (myocytes, vascular endothelial cells and smooth muscle cells) can be derived and used for cardiac repair in case of heart injury. It has been proposed that the clinical benefit of stem cells may arise from secreted proteins that mediate regeneration in a paracrine/autocrine manner. To be able to track the regulatory pathway on a molecular basis, utilization of proteomics in stem cell research is essential. Proteomics offers a tool that can address questions regarding stem cell response to disease/injury.

Analysis of protein isoforms: can we do it better?

Protein isoforms/splice variants can play important roles in various biological processes and can potentially be used as biomarkers or therapeutic targets/mediators. Thus, there is a need for efficient and, importantly, accurate methods to distinguish and quantify specific protein isoforms. Since protein isoforms can share a high percentage of amino acid sequence homology and dramatically differ in their cellular concentration, the task for accuracy and efficiency in methodology and instrumentation is challenging. The analysis of intact proteins has been perceived to provide a more accurate and complete result for isoform identification/quantification in comparison to analysis of the corresponding peptides that arise from protein enzymatic digestion. Recently, novel approaches have been explored and developed that can possess the accuracy and reliability important for protein isoform differentiation and isoform‐specific peptide targeting. In this review, we discuss the recent development in methodology and instrumentation for enhanced detection of protein isoforms as well as the examples of their biological importance.

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

Interaction among different types of cells is a common response of the heart to injury. Interplay between various cell types constituting the heart or the vasculature occurs either through direct cell contact (eg, gap junctions or adhesion molecules) or via paracrine and autocrine action. Cardiac myocytes and fibroblasts, the primary cells that comprise the heart, as well as resident cardiac stem cells (CSCs), respond to changes in their local environment, whereas it is the cell surface that translates the extracellular signal into a cellular response through direct or indirect interactions. The main site of this response is the cell-environment interface, which includes both cell surface proteins and proteins that are secreted by cells as a reaction to these changes. Various methods have been developed to identify these subproteomes and clarify protein alterations in vivo. The ability to target and define the proteins responsible for the repair process could lead to improved therapy and to the development of potential biomarkers for disease diagnosis and prognosis. In this article, we will discuss the technical issues related to identification of 1 of these subproteomes, ie, the proteins secreted by cardiac cells as a response to the alteration in their environment caused by cardiovascular diseases. The proteins secreted by a particular type of cell, secretomes, play important roles in the regulation of many physiological processes via paracrine/autocrine mechanisms, and they are of increasing interest as potential biomarkers and therapeutic targets in diseases. The proteins released by cells into conditioned media in vitro have been studied to better understand pathological conditions and mechanisms in vivo. For damaged myocardium, current therapeutic approaches are limited, because postnatal cardiac myocytes have minimum or no regenerative capacity. Thus, the ability to regenerate the myocardium by use of cell therapy has potential, and several experimental and clinical studies have demonstrated …

Analysis of protein composition of rabbit aqueous humor following two different cataract surgery incision procedures using 2-DE and LC-MS/MS

BackgroundThe aqueous humor (AH), a liquid of the anterior and posterior chamber of the eye, comprises many proteins with various roles and important biological functions. Many of these proteins have not been identified yet and their functions in AH are still unknown. Recently, our laboratory published the protein database of AH obtained from healthy rabbits which expanded known protein identifications by 65%. Our present study extends our previous work and analyses AH following two types of cataract surgery incision procedures (clear corneal and limbal incisions) by using two dimensional gel electrophoresis (2-DE) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Although both incision protocols are commonly used during cataract surgeries, the difference in protein composition and their release into AH following each surgery has never been systematically compared and remains unclear. The first step, which is the focus of this work, is to assess the scale of the protein change, at which time does maximum release occurs and when possible, to identify protein changes.ResultsSamples of AH obtained prior to surgery and at different time points (0.5, 2, 12, 24 and 48 hours) following surgery (n = 3/protocol) underwent protein concentration determination, 2-DE and LC-MS/MS. There was a large (9.7 to 31.2 mg/mL) and rapid (~0.5 hour) influx of proteins into AH following either incision with a return to baseline quantities after 12 hours and 24 hours for clear corneal and limbal incision, respectively. We identified 80 non-redundant proteins, and compared to our previous study on healthy AH, 67.5% of proteins were found to be surgery-specific. In addition, 51% of those proteins have been found either in clear corneal (20%) or limbal incision (31%) samples.ConclusionsOur results imply that a mechanism of protein release into AH after surgery is a global response to the surgery rather than increase in amount of protective proteins found in healthy AH and a mechanism of protein release for each type of incision procedure could be different. Although the total protein concentration was increased (at 0.5 and 2 hour time points and between types of surgery) many of 2-DE protein spots were similar based on 2-DE and MS analyses, and only a small number of protein spots changed with either the time points or surgical conditions (0.4 -1.9%). This suggests that the high protein content is due to an increase in the concentration of the same proteins with only a few unique proteins being altered per time point and with the different surgery type. This is the first report on the comparison of AH protein composition following two different cataract surgery procedures and it establishes the basis for better understanding of protein release into AH during events such as cataract surgery or other possible intervention to the eyes.

Optimized Method for Identification of the Proteomes Secreted by Cardiac Cells

In the past, various studies using different methods have been carried out to analyze proteins secreted by cells. There are several crucial steps that have to be followed to ensure successful secreted proteome detection and identification. Simultaneously with the optimization of the experimental conditions for various cell type culturing and subsequent cell conditioning to obtain conditioned medium with secreted proteins in vitro, the analytical separation methods for fractionation of complex protein mixture and mass spectrometry for protein identification are of high importance. The separation methods primarily used are either gel-based (e.g., 1-DE and 2-DE) or gel-free methods (e.g., liquid chromatography and capillary electrophoresis). Here we outline an optimized protocol for the preparation and analysis of conditioned medium containing proteins secreted by neonatal cardiac myocytes by using reversed-phase liquid chromatography (RPLC) followed by tandem mass spectrometry (LC-MS/MS). Although optimized for neonatal cardiac myocytes, the general steps described in the following chapter can be adapted to other cell types as well.

Cardiovascular proteomics: Assessment of protein post-translational modifications

Proteomics is the study of proteins and it employs methods that characterize their diversity. It is based on detection, identification, and quantification of proteins, isoforms, and post-translational modifications (PTMs). Proteomics is used to study how the proteome is altered in response to changes in the environment, changes that allow the cell to adapt to acute and ongoing chronic stimuli. The stimuli almost always result in PTMs of specific proteins and a subset of these changes can result in rapid and dynamic changes to the cells. In the heart, the most commonly PTM studied is phosphorylation, yet, there is increasing recognition of the role of S-nitrosation, acetylation, and O-GlcNAcylation. With technology development, proteomics has become a powerful identification tool for discovery of new markers for prognosis, diagnosis, and therapy. This chapter outlines the basic analytical protein biochemical techniques used in proteomics with subsequent examples of biological application to analysis of cardiac subproteomes and PTMs.