High-Throughput LC-MS/MS Proteomic Analysis of a Mouse Model of Mesiotemporal Lobe Epilepsy Predicts Microglial Activation Underlying Disease Development.

Uncovering the molecular mechanisms of mesiotemporal lobe epilepsy (MTLE) is critical to identify therapeutic targets. In this study, we performed global protein expression analysis of a kainic acid (KA) MTLE mouse model at various time-points (1, 3, and 30 days post-KA injection -dpi), representing specific stages of the syndrome. High-resolution liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), in combination with label-free protein quantification using three processing approaches for quantification, was applied. Following comparison of KA versus NaCl-injected mice, 22, 53, and 175 proteins were differentially (statistically significant) expressed at 1, 3 and 30dpi, respectively, according to all three quantification approaches. Selected findings were confirmed by multiple reaction monitoring LC-MS/MS. As a positive control, the astrocyte marker GFAP was found to be upregulated (3dpi: 1.9 fold; 30dpi: 12.5 fold), also verified by IHC. The results collectively suggest that impairment in synaptic transmission occurs even right after initial status epilepticus (1dpi), with neurodegeneration becoming more extensive during epileptogenesis (3dpi) and sustained at the chronic phase (30dpi), where also extensive glial- and astrocyte-mediated inflammation is evident. This molecular profile is in line with observed phenotypic changes in human MTLE, providing the basis for future studies on new molecular targets for the disease.

Profilin 1 is a potential biomarker for bladder cancer aggressiveness.

Of the most important clinical needs for bladder cancer (BC) management is the identification of biomarkers for disease aggressiveness. Urine is a "gold mine" for biomarker discovery, nevertheless, with multiple proteins being in low amounts, urine proteomics becomes challenging. In the present study we applied a fractionation strategy of urinary proteins based on the use of immobilized metal affinity chromatography for the discovery of biomarkers for aggressive BC. Urine samples from patients with non invasive (two pools) and invasive (two pools) BC were subjected to immobilized metal affinity chromatography fractionation and eluted proteins analyzed by 1D-SDS-PAGE, band excision and liquid chromatography tandem MS. Among the identified proteins, multiple corresponded to proteins with affinity for metals and/or reported to be phosphorylated and included proteins with demonstrated association with BC such as MMP9, fibrinogen forms, and clusterin. In agreement to the immobilized metal affinity chromatography results, aminopeptidase N, profilin 1, and myeloblastin were further found to be differentially expressed in urine from patients with invasive compared with non invasive BC and benign controls, by Western blot or Elisa analysis, nevertheless exhibiting high interindividual variability. By tissue microarray analysis, profilin 1 was found to have a marked decrease of expression in the epithelial cells of the invasive (T2+) versus high risk non invasive (T1G3) tumors with occasional expression in stroma; importantly, this pattern strongly correlated with poor prognosis and increased mortality. The functional relevance of profilin 1 was investigated in the T24 BC cells where blockage of the protein by the use of antibodies resulted in decreased cell motility with concomitant decrease in actin polymerization. Collectively, our study involves the application of a fractionation method of urinary proteins and as one main result of this analysis reveals the association of profilin 1 with BC paving the way for its further investigation in BC stratification.

Improving peptide relative quantification in MALDI-TOF MS for biomarker assessment.

Proteomic profiling by MALDI-TOF MS presents various advantages (speed of analysis, ease of use, relatively low cost, sensitivity, tolerance against detergents and contaminants, and possibility of automation) and is being currently used in many applications (e.g. peptide/protein identification and quantification, biomarker discovery, and imaging MS). Earlier studies by many groups indicated that moderate reproducibility in relative peptide quantification is a major limitation of MALDI-TOF MS. In the present work, we examined and demonstrate a clear effect, in cases apparently random, of sample dilution in complex samples (urine) on the relative quantification of peptides by MALDI-TOF MS. Results indicate that in urine relative abundance of peptides cannot be assessed with confidence based on a single MALDI-TOF MS spectrum. To account for this issue, we developed and propose a novel method of determining the relative abundance of peptides, taking into account that peptides have individual linear quantification ranges in relation to sample dilution. We developed an algorithm that calculates the range of dilutions at which each peptide responds in a linear manner and normalizes the received peptide intensity values accordingly. This concept was successfully applied to a set of urine samples from patients diagnosed with diabetes presenting normoalbuminuria (controls) and macroalbuminuria (cases).

Therapeutic potential of a distinct population of human amniotic fluid mesenchymal stem cells and their secreted molecules in mice with acute hepatic failure.

BACKGROUND: There is increasing interest in the therapeutic potential of human mesenchymal stem cells (hMSCs), especially in diseases such as acute hepatic failure (AHF) that are predominantly caused by a variety of drugs and viruses. In previous studies, a distinct population termed human spindle-shaped MSCs were isolated and expanded from second trimester amniotic fluid (AF-MSCs) and characterised based on their phenotype, pluripotency and differentiation potential. METHODS: AF-MSCs, hepatic progenitor-like (HPL) cells and hepatocyte-like (HL) cells derived from AF-MSCs were transplanted into CCl4-injured NOD/SCID mice with the AHF phenotype in order to evaluate their therapeutic potential. Conditioned medium (CM) derived from AF-MSCs or HPL cells was then delivered intrahepatically in order to determine whether the engraftment of the cells or their secreted molecules are the most important agents for liver repair. RESULTS: Both HPL cells and AF-MSCs were incorporated into CCl(4)-injured livers; HPL cell transplantation had a greater therapeutic effect. In contrast, HL cells failed to engraft and contribute to recovery. In addition, HPL-CM was found to be more efficient than CM derived from AF-MSCs in treatment of the liver. Proteome profile analysis of HPL-CM indicated the presence of anti-inflammatory factors such as interleukins IL-10, IL-1ra, IL-13 and IL-27 which may induce liver recovery. Blocking studies of IL-10 secretion from HPL cells confirmed the therapeutic significance of this cytokine in the AHF mouse model. CONCLUSIONS: Human spindle-shaped AF-MSCs or HPL cells might be valuable tools to induce liver repair and support liver function by cell transplantation. More importantly, the factors they release may also play an important role in cell treatment in diseases of the liver.

In vitro and in vivo properties of distinct populations of amniotic fluid mesenchymal progenitor cells.

Human mesenchymal progenitor cells (MPCs) are considered to be of great promise for use in tissue repair and regenerative medicine. MPCs represent multipotent adherent cells, able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes or chondrocytes. Recently, we identified and characterized human second trimester amniotic fluid (AF) as a novel source of MPCs. Herein, we found that early colonies of AF-MPCs consisted of two morphologically distinct adherent cell types, termed as spindle-shaped (SS) and round-shaped (RS). A detailed analysis of these two populations showed that SS-AF-MPCs expressed CD90 antigen in a higher level and exhibited a greater proliferation and differentiation potential. To characterize better the molecular identity of these two populations, we have generated a comparative proteomic map of SS-AF-MPCs and RS-AF-MPCs, identifying 25 differentially expressed proteins and 10 proteins uniquely expressed in RS-AF-MPCs. Furthermore, SS-AF-MPCs exhibited significantly higher migration ability on extracellular matrices, such as fibronectin and laminin in vitro, compared to RS-AF-MPCs and thus we further evaluated SS-AF-MPCs for potential use as therapeutic tools in vivo. Therefore, we tested whether GFP-lentiviral transduced SS-AF-MPCs retained their stem cell identity, proliferation and differentiation potential. GFP-SS-AF-MPCs were then successfully delivered into immunosuppressed mice, distributed in different tissues and survived longterm in vivo. In summary, these results demonstrated that AF-MPCs consisted of at least two different MPC populations. In addition, SS-AF-MPCs, isolated based on their colony morphology and CD90 expression, represented the only MPC population that can be expanded easily in culture and used as an efficient tool for future in vivo therapeutic applications.

Analysis of secreted proteins for the study of bladder cancer cell aggressiveness.

Secreted proteins play a key role in cell signaling, communication, and migration. We recently described the development of an aggressive variant (T24M) of the bladder cancer cell line T24. Using this cell line model, the objective of our work was the identification of secreted proteins involved in the acquisition of the aggressive phenotype. Using in vitro assays, we demonstrate that conditioned media of the T24M cells promote motility of the parental less aggressive T24 cells. Proteomic analysis of cell culture conditioned media by the use of 2-dimensional gel electrophoresis coupled to MALDI TOF MS and LC-MS approaches resulted in enrichment and detection of multiple classical extracellular and secreted proteins such as fibronectin, cystatin, fibrillin, fibulin, interleukin 6, etc. Comparison of the secretome of the T24 and T24M cells indicated differences in proteins with potential involvement in the mechanisms of cell aggressiveness including SPARC, tPA, and clusterin. These findings were further confirmed by Western blot analysis. In the case of SPARC, further studies involving transwell assays indicated that blockage of the protein in the presence of SPARC-specific Abs results in decreased cell motility. Collectively, our study provides a 2DE-based comprehensive analysis of bladder cancer cell secretome. The results indicate various secreted proteins with potential involvement in bladder cancer cell aggressiveness and more specifically provide initial evidence for special role of SPARC in bladder cancer cell motility and invasiveness.

Chromosomal and proteome analysis of a new T24-based cell line model for aggressive bladder cancer.

Cell line models aid in understanding cancer aggressiveness. The aim of this study was the establishment of a metastatic variant (T24M) of the T24 bladder cancer cell line and its initial characterization at chromosomal and proteomic levels. T24M were spontaneously developed in mice from T24 cells, following cycles of subcutaneous injections and culture in vitro. Transwell migration assays and injections in mice revealed increased migration and tumorigenic properties of T24M compared to the T24 cells. Cytogenetic analysis demonstrated that T24M retained several karyotypic characteristics of the parental cells and also acquired novel chromosomal aberrations related to aggressive bladder cancer. Proteomic analysis of the T24 and T24M cells by 2-DE and MS led to the generation of their 2-DE proteomic map and revealed differences in multiple proteins. These include proteases of the lysosomal and proteasome degradation pathways, mitochondrial and cytoskeletal proteins. The 2-DE findings were confirmed by immunoblotting of cell lysates and immunohistochemistry of bladder cancer tissue sections for cathepsin D and activity assays for proteasome. Collectively, our results suggest that the T24M cells reflect many known chromosomal and proteomic aberrations encountered in aggressive bladder cancers but also provide access to novel findings with potentially clinical applications.

Development and Validation of Multiple Reaction Monitoring (MRM) Assays for Clinical Applications.

Selected/multiple reaction monitoring-mass spectrometry (SRM/MRM) is an analytical method that is frequently combined to the use of stable isotope-labeled standard (SIS) peptides for absolute protein quantification. The application of SRM/MRM is a relatively recent development in the proteomics field for analysis of biological samples (plasma, urine, cell/tissue lysates) targeting to a large extent biomarker validation. Although MRM generally by-passes the use of antibodies (being linked to sub-optimal assay specificity in many cases), bioanalytical validation of MRM protocols has not been robustly applied because of sensitivity issues, in contrary to antibody-based methods. In this chapter, we will discuss the points that should be addressed for MRM method development in clinical proteomics applications.

Proteomics based identification of KDM5 histone demethylases associated with cardiovascular disease.

BACKGROUND: The increased prevalence of cardiovascular disease (CVD) indicates a demand for novel therapeutic approaches. Proteome analysis of vascular tissues from animal models and humans with CVD could lead to the identification of novel druggable targets. METHODS: LC-MS/MS analysis of thoracic aortas from three mouse models of non-diabetic and diabetic (streptozotocin (STZ)-induced) atherosclerosis followed by bioinformatics/pathway analysis was performed. Selected findings were confirmed by proteomics analysis of human vessels from patients with CVD as well as in vitro studies (migration, proliferation, angiogenesis assays) using endothelial (HUVEC) cells. FINDINGS: Comparative tissue proteomics of low density lipoprotein receptor deficient (Ldlr-/-) and diabetic Ldlr-/- (Ldlr-/-STZ) with wild type (WT) animals led to the identification of 284 differentially expressed proteins in both models. Among them, 177 proteins were also differentially expressed in diabetic apolipoprotein E deficient (ApoE-/-STZ) mice, suggesting expression changes associated with atherosclerosis independent of the model used. These proteins recapitulated the hallmarks of atherosclerosis. Comparison of these findings with differentially expressed proteins in human vessels with CVD enabled shortlisting of six commonly dysregulated proteins. Among them, lysine-specific demethylase 5D (KDM5D) exhibited pronounced overexpression accompanied by a reduction in the protein levels of its substrate, the trimethylated lysine 4 of histone H3 (H3K4me3), in patients with CVD. Functional interference studies applying a KDM5 inhibitor on HUVEC reduced cell proliferation, migration and tube-forming ability in vitro. INTERPRETATION: This high-throughput proteomics strategy identified KDM5 histone demethylases being potentially involved in CVD, possibly by affecting H3K4 methylation. FUND: [SysVasc, HEALTH-2013 603288], [ERA-CVD PROACT: ANR-17-ECVD-0006, 01KL1805], [FRM, DEQ20170336759].

Molecular and proteomic characterization of human mesenchymal stem cells derived from amniotic fluid: comparison to bone marrow mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) constitute a population of multipotent adherent cells able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes, or chondrocytes. So far, the most common source of MSCs has been the bone marrow (BM); however BM-MSC harvesting and processing exhibits major drawbacks and limitations. Thus, identification and characterization of alternative sources of MSCs are of great importance. In the present study, we isolated and expanded fetal MSCs from second-trimester amniotic fluid (AF). We documented that these cells are of embryonic origin, can differentiate under appropriate conditions into cell types derived from all three germ layers, and express the pluripotency marker Oct-4, the human Nanog protein, and the stage-specific embryonic antigen-4 (SSEA-4). Furthermore, we systematically tested the immunophenotype of cultured MSCs by flow cytometry analysis using a wide variety of markers. Direct comparison of this phenotype to the one derived from cultured BM-MSCs demonstrated that cultured MSCs from both sources exhibit similar expression patterns. Using the two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) approach, we have generated for the first time the protein map of cultured AF-MSCs by identifying 261 proteins, and we compared it directly to that of cultured BM-MSCs. The functional pattern of the identified proteins from both sources was similar. However, cultured AF-MSCs displayed a number of unique proteins related to proliferation and primitive phenotype, which may confer to the distinct features of the two types. Considering the easy access to this new cell source and the yield of expanded MSCs for stem cell research, AF may provide an excellent source of MSCs both for basic research and for potential therapeutic applications.

MALDI-imaging mass spectrometry on tissues.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-profiling and imaging mass spectrometry (MSI) are promising technologies for measuring hundreds of different molecules directly on tissues. For instance, small molecules, drugs and their metabolites, endogenous lipids, carbohydrates and complex peptides/proteins can be measured at the same time. In the most advanced instruments, it is achieved without significant disruption of sample integrity. MSI is a unique approach for assessing the spatial distribution of molecules using graphical multidimensional maps of their constituent analytes, which may for instance be correlated with histopathological alterations in patient tissues. MALDI-TOF-MSI technology has been implemented in hospitals of several countries, where it is routinely used for quick pathogen(s) identification, a task formerly accomplished by laborious and expensive DNA/RNA-based PCR (polymerase chain reaction) screening.In this chapter, we describe how MSI is performed, what is required from the researcher, the instrument vendors and finally what can be achieved with MSI. We restrict our descriptions only to MALDI-MSI although several other MS techniques of ionization can easily be linked to MSI.

Capillary zone electrophoresis on-line coupled to mass spectrometry: A perspective application for clinical proteomics.

Clinical proteomics, a rapidly growing field, intends to use specific diagnostic proteomic/peptidomic markers for initial diagnosis or prognosis of the progression of various diseases. Analyses of disease-associated markers in defined biological samples can provide valuable molecular diagnostic information for these diseases. This approach relies on sensitive and highly standardized modern analytical techniques. In the recent years, one of these technologies, CZE online coupled to MS (CZE-MS), has been increasingly used for the detection of peptide biomarkers (<20 kDa) in body fluids such as urine. This review presents the most relevant urinary proteomic studies addressing the application of CZE-MS in clinically relevant biomarker research between the years 2006 and 2014.

Comparison of higher energy collisional dissociation and collision-induced dissociation MS/MS sequencing methods for identification of naturally occurring peptides in human urine.

PURPOSE: The aim of this study is to determine the best fragmentation method for sequence identification of naturally occurring urinary peptides in the field of clinical proteomics. EXPERIMENTAL DESIGN: We used LC-MS/MS analysis of urine samples to determine the analytical performance of higher energy collisional dissociation (HCD), CID with high and low resolution MS/MS for the identification of naturally occurring peptides in the low molecular weight urinary proteome. RESULTS: HCD and CID high-resolution generated a 22% error rate in peptide sequence identifications. CID low-resolution showed significantly higher error rates (37%). Excluding the error rate (i.e rejection of cysteine-containing peptides), we observed a higher degree of overlap between HCD and CID high resolution for identification of peptide sequences of rank 1 and cross-correlation ≥ 1.9 (262 peptide sequences) compared to CID low (208 peptide sequences with HCD and 192 peptide sequences with CID high). Reproducibility of detected peptides in three out of the five replicates was also higher in HCD and CID high in relation to CID low resolution. CONCLUSION AND CLINICAL RELEVANCE: Our data demonstrated that HCD and CID high-resolution performed with better accuracy and reproducibility than CID low resolution in respect to the identification of naturally occurring urinary peptide sequences.

Sox2 suppression by miR-21 governs human mesenchymal stem cell properties.

MicroRNAs (miRNAs) have recently been shown to act as regulatory signals for maintaining stemness and for determining the fate of adult and fetal stem cells, such as human mesenchymal stem cells (hMSCs). hMSCs constitute a population of multipotent stem cells that can be expanded easily in culture and are able to differentiate into many lineages. We have isolated two subpopulations of fetal mesenchymal stem cells (MSCs) from amniotic fluid (AF) known as spindle-shaped (SS) and round-shaped (RS) cells and characterized them on the basis of their phenotypes, pluripotency, proliferation rates, and differentiation potentials. In this study, we analyzed the miRNA profile of MSCs derived from AF, bone marrow (BM), and umbilical cord blood (UCB). We initially identified 67 different miRNAs that were expressed in all three types of MSCs but at different levels, depending on the source. A more detailed analysis revealed that miR-21 was expressed at higher levels in RS-AF-MSCs and BM-MSCs compared with SS-AF-MSCs. We further demonstrated for the first time a direct interaction between miR-21 and the pluripotency marker Sox2. The induction of miR-21 strongly inhibited Sox2 expression in SS-AF-MSCs, resulting in reduced clonogenic and proliferative potential and cell cycle arrest. Strikingly, the opposite effect was observed upon miR-21 inhibition in RS-AF-MSCs and BM-MSCs, which led to an enhanced proliferation rate. Finally, miR-21 induction accelerated osteogenesis and impaired adipogenesis and chondrogenesis in SS-AF-MSCs. Therefore, these findings suggest that miR-21 might specifically function by regulating Sox2 expression in human MSCs and might also act as a key molecule determining MSC proliferation and differentiation.

Fetal mesenchymal stem cells in cancer therapy.

There is compelling evidence that mesenchymal stem cells (MSCs) can be utilized as delivery vehicles for cancer therapeutics. During the last decade, bone marrow MSCs have been used as delivery vehicles for the local production of therapeutic proteins in multiple tumor types, taking advantage of their innate tropism to the tumor site and their low immunogenicity. More recently, MSCs have been isolated from fetal tissues during gestation or after birth. Fetal MSCs derived from amniotic fluid, amniotic membrane, umbilical cord matrix (Wharton's jelly) and umbilical cord blood are more advantageous than adult MSCs, as they can be isolated noninvasively in large numbers without the ethical reservations associated with embryo research. Several studies have documented that fetal MSCs harbor a therapeutic potential in cancer treatment, as they can home to the tumor site and reduce tumor burden. This natural tumor tropism together with their low immunogenicity renders fetal MSCs as powerful therapeutic tools in gene therapy-based cancer therapeutic schemes. This review summarizes various approaches where the tumor-homing capacity of fetal MSCs has been employed for the localized delivery of anti-tumor therapeutic agents.

High-resolution proteome/peptidome analysis of body fluids by capillary electrophoresis coupled with MS.

The analysis of proteins and peptides in biological fluids is becoming more important as they are potential sources of diagnostic biomarkers of disease. The complexity of body fluids is such that no single technique can both identify and quantify all the constituents present. It therefore requires pre-concentration of the compounds of interest and hyphenated techniques like capillary electrophoresis (CE) or high-performance liquid chromatography (HPLC) coupled to a mass spectrometer (MS) to provide the separation, identification, and quantification of the proteins and peptides under investigation. Here we provide protocols for sample preparation of various body fluids, namely, urine, plasma, and cerebrospinal fluid, and how to obtain maximum sensitivity and selectivity of the small proteins and peptides under investigation by CE-MS.

Human amniotic fluid-derived mesenchymal stem cells as therapeutic vehicles: a novel approach for the treatment of bladder cancer.

Recent studies support cell-based therapies for cancer treatment. An advantageous cell type for such therapeutic schemes are the mesenchymal stem cells (MSCs) that can be easily propagated in culture, genetically modified to express therapeutic proteins, and exhibit an innate tropism to solid tumors in vivo. Recently, we successfully isolated and expanded MSCs from second-trimester amniotic fluid (AF-MSCs). The main characteristic of AF-MSCs is their efficient and rapid expansion in vitro. Herein, we investigated the AF-MSCs tropism and capability to transport interferon beta (IFNß) to the region of neoplasia in a bladder tumor model. To this end, we used the T24M bladder cancer cell line, previously generated from our studies, and developed a disease progression model in immunosuppressed mice, that can recapitulate the molecular events of bladder carcinogenesis. Our results documented that AF-MSCs exhibited high motility, when migrated either to T24M cells or to T24M-conditioned medium, and we further identified and studied the secreted factors which may trigger these enhanced migratory properties. Further, lentivirus-transduced AF-MSCs, expressing green fluorescent protein (GFP) or IFNß, were intravenously administered to T24M tumor-bearing animals at multiple doses to examine their therapeutic effect. GFP- and IFNß-AF-MSCs successfully migrated and colonized at the tumor site. Notably, significant inhibition of tumor growth as well as prolonged survival of mice were observed in the presence of IFNß-AF-MSCs. Collectively, these results document the great potential of AF-MSCs as anti-cancer vehicles, implemented by the targeting of the tumor site and further facilitated by their high proliferation rate and expansion efficiency in culture.