Characterization of Human B Cell Hematological Malignancies Using Protein-Based Approaches.

The maturation of B cells is a complex, multi-step process. During B cell differentiation, errors can occur, leading to the emergence of aberrant versions of B cells that, finally, constitute a malignant tumor. These B cell malignancies are classified into three main groups: leukemias, myelomas, and lymphomas, the latter being the most heterogeneous type. Since their discovery, multiple biological studies have been performed to characterize these diseases, aiming to define their specific features and determine potential biomarkers for diagnosis, stratification, and prognosis. The rise of advanced -omics approaches has significantly contributed to this end. Notably, proteomics strategies appear as promising tools to comprehensively profile the final molecular effector of these cells. In this narrative review, we first introduce the main B cell malignancies together with the most relevant proteomics approaches. Then, we describe the core studies conducted in the field and their main findings and, finally, we evaluate the advantages and drawbacks of flow cytometry, mass cytometry, and mass spectrometry for the profiling of human B cell disorders.

Toxicological Profiling and Long-Term Effects of Bare, PEGylated- and Galacto-Oligosaccharide-Functionalized Mesoporous Silica Nanoparticles.

Mesoporous silica nanoparticles (MSNs) are amongst the most used nanoparticles in biomedicine. However, the potentially toxic effects of MSNs have not yet been fully evaluated, being a controversial matter in research. In this study, bare MSNs, PEGylated MSNs (MSNs-PEG), and galacto-oligosaccharide-functionalized MSNs (MSNs-GAL) are synthesized and characterized to assess their genotoxicity and transforming ability on human lung epithelial BEAS-2B cells in short- (48 h) and long-term (8 weeks) exposure scenarios. Initial short-term treatments show a dose-dependent increase in genotoxicity for MSNs-PEG-treated cells but not oxidative DNA damage for MSNs, MSNs-PEG, or for MSNs-GAL. In addition, after 8 weeks of continuous exposure, neither induced genotoxic nor oxidative DNA is observed. Nevertheless, long-term treatment with MSNs-PEG and MSNs-GAL, but not bare MSNs, induces cell transformation features, as evidenced by the cell's enhanced ability to grow independently of anchorage, to migrate, and to invade. Further, the secretome from cells treated with MSNs and MSNs-GAL, but not MSNs-PEG, shows certain tumor-promoting abilities, increasing the number and size of HeLa cell colonies formed in the indirect soft-agar assay. These results show that MSNs, specifically the functionalized ones, provoke some measurable adverse effects linked to tumorigenesis. These effects are in the order of other nanomaterials, such as carbon nanotubes or cerium dioxide nanoparticles, but they are lower than those provoked by some approved drugs, such as doxorubicin or dexamethasone.

Comprehensive and systematic characterization of multi-functionalized cisplatin nano-conjugate: from the chemistry and proteomic biocompatibility to the animal model.

BACKGROUND: Nowadays, nanoparticles (NPs) have evolved as multifunctional systems combining different custom anchorages which opens a wide range of applications in biomedical research. Thus, their pharmacological involvements require more comprehensive analysis and novel nanodrugs should be characterized by both chemically and biological point of view. Within the wide variety of biocompatible nanosystems, iron oxide nanoparticles (IONPs) present mostly of the required features which make them suitable for multifunctional NPs with many biopharmaceutical applications. RESULTS: Cisplatin-IONPs and different functionalization stages have been broadly evaluated. The potential application of these nanodrugs in onco-therapies has been assessed by studying in vitro biocompatibility (interactions with environment) by proteomics characterization the determination of protein corona in different proximal fluids (human plasma, rabbit plasma and fetal bovine serum),. Moreover, protein labeling and LC-MS/MS analysis provided more than 4000 proteins de novo synthetized as consequence of the nanodrugs presence defending cell signaling in different tumor cell types (data available via ProteomeXchanges with identified PXD026615). Further in vivo studies have provided a more integrative view of the biopharmaceutical perspectives of IONPs. CONCLUSIONS: Pharmacological proteomic profile different behavior between species and different affinity of protein coating layers (soft and hard corona). Also, intracellular signaling exposed differences between tumor cell lines studied. First approaches in animal model reveal the potential of theses NPs as drug delivery vehicles and confirm cisplatin compounds as strengthened antitumoral agents.

Proteomics for Low Cell Numbers: How to Optimize the Sample Preparation Workflow for Mass Spectrometry Analysis.

Nowadays, massive genomics and transcriptomics data can be generated at the single-cell level. However, proteomics in this setting is still a big challenge. Despite the great improvements in sensitivity and performance of mass spectrometry instruments and the better knowledge on sample preparation processing, it is widely acknowledged that multistep proteomics workflows may lead to substantial sample loss, especially when working with paucicellular samples. Still, in clinical fields, frequently limited sample amounts are available for downstream analysis, thereby hampering comprehensive characterization at protein level. To aim at better protein and peptide recoveries, we compare existing and novel approaches in the multistep sample preparation protocols for mass spectrometry studies, from sample collection, cell lysis, protein quantification, and electrophoresis/staining to protein digestion, peptide recovery, and LC-MS/MS instruments. From this critical evaluation, we conclude that the recent innovations and technologies, together with high quality management of samples, make proteomics on paucicellular samples possible, which will have immediate impact for the proteomics community.

A Novel Cytotoxic Conjugate Derived from the Natural Product Podophyllotoxin as a Direct-Target Protein Dual Inhibitor.

Natural products are the ideal basis for the design of novel efficient molecular entities. Podophyllotoxin, a naturally occurring cyclolignan, is an example of natural product which displays a high versatility from a biological activity point of view. Based on its unique chemical structure, different derivatives have been synthesized presenting the original antitumoral properties associated with the compound, i.e., the tubulin polymerization inhibition and arising anti-topoisomerase II activity from structural modifications on the cyclolignan skeleton. In this report, we present a novel conjugate or hybrid which chemically combines both biological activities in one single molecule. Chemical design has been planned based in our lead compound, podophyllic aldehyde, as an inhibitor of tubulin polymerization, and in etoposide, an approved antitumoral drug targeting topoisomerase II. The cytotoxicity and selectivity of the novel synthetized hybrid has been evaluated in several cell lines of different solid tumors. In addition, these dual functional effects of the novel compound have been also evaluated by molecular docking approaches.

Screening and Validation of Novel Biomarkers in Osteoarticular Pathologies by Comprehensive Combination of Protein Array Technologies.

Osteoarthritis (OA) is one of the most prevalent articular diseases. The identification of proteins closely associated with the diagnosis, progression, prognosis, and treatment response is dramatically required for this pathology. In this work, differential serum protein profiles have been identified in OA and rheumatoid arthritis (RA) by antibody arrays containing 151 antibodies against 121 antigens in a cohort of 36 samples. Then the identified differential serum protein profiles have been validated in a larger cohort of 282 samples. The overall immunoreactivity is higher in the pathological situations in comparison with the controls. Several proteins have been identified as biomarker candidates for OA and RA. Most of these biomarker candidates are proteins related to inflammatory response, lipid metabolism, or bone and extracellular matrix formation, degradation, or remodeling.

Functional proteomic insights in B-cell chronic lymphocytic leukemia.

INTRODUCTION: B cell chronic lymphocytic leukemia (B-CLL) is a hematological malignancy considered as the most common leukemia in the Western world. The understanding of B cell differentiation is crucial for the diagnosis, prognosis, and treatment of the disease. Areas covered: In this review, B-cell ontogeny and its relation with the CLL development, in combination with the proteomic approaches which could provide a deep characterization of the disease through the characterization of the cellular signaling pathways involved in the pathological cells is described. Expert commentary: Although conventional strategies (genome sequencing, morphology assays, and immunophenotyping by flow cytometry and/or immunochemistry) have allowed the establishment of the disease stage based on different parameters, it is still necessary to utilize novel approaches (e.g., proteomics) that have the potential to simultaneously analyze thousands of molecules to improve understanding of CLL.

Comprehensive and Systematic Analysis of the Immunocompatibility of Polyelectrolyte Capsules.

The immunocompability of polyelectrolyte capsules synthesized by layer-by-layer deposition has been investigated. Capsules of different architecture and composed of either non-degradable or biodegradable polymers, with either positively or negatively charged outer surface, and with micrometer size, have been used, and the capsule uptake by different cell lines has been studied and quantified. Immunocompatibility studies were performed with peripheral blood mononuclear cells (PBMCs). Data demonstrate that incubation with capsules, at concentrations relevant for practical applications, did not result in a reduced viability of cells, as it did not show an increased apoptosis. Presence of capsules also did not result in an increased expression of TNF-α, as detected with antibody staining, as well as at mRNA level. It also did not result in increased expression of IL-6, as detected at mRNA level. These results indicate that the polyelectrolyte capsules used in this study are immunocompatible.

Self-Regulated Glucose-Sensitive Neoglycoenzyme-Capped Mesoporous Silica Nanoparticles for Insulin Delivery.

We describe herein the preparation of glucose-sensitive capped mesoporous silica nanoparticles for insulin delivery. The new material consists of an expanded-pore nanometric silica support grafted with 1-propyl-1-H-benzimidazole groups, loaded with fluorescein isothiocyanate-labeled insulin (FITC-Ins) and capped by the formation of inclusion complexes between cyclodextrin-modified glucose oxidase (CD-GOx) and the benzimidazole groups grafted on the mesoporous support. Insulin delivery from the gated material in simulated blood plasma was assessed upon addition of glucose. Glucose is transformed by GOx into gluconic acid, which promoted the dethreading of the benzimidazole-CD-GOx inclusion complexes, allowing cargo release. Small quantities of this support would be needed to release the amount of insulin necessary to decrease diabetic blood glucose concentrations to regular levels.

Enzyme-Controlled Nanodevice for Acetylcholine-Triggered Cargo Delivery Based on Janus Au-Mesoporous Silica Nanoparticles.

This work reports a new gated nanodevice for acetylcholine-triggered cargo delivery. We prepared and characterized Janus Au-mesoporous silica nanoparticles functionalized with acetylcholinesterase on the Au face and with supramolecular ß-cyclodextrin:benzimidazole inclusion complexes as caps on the mesoporous silica face. The nanodevice is able to selectively deliver the cargo in the presence of acetylcholine via enzyme-mediated acetylcholine hydrolysis, locally lowering the pH and opening the supramolecular gate. Given the key role played by ACh and its relation with Parkinson's disease and other nervous system diseases, we believe that these findings could help design new therapeutic strategies.

Proteomic Biomarker Identification in Cerebrospinal Fluid for Leptomeningeal Metastases with Neurological Complications.

Leptomeningeal metastases (LM) from solid tumours, lymphoma and leukaemia are characterized by multifocal neurological deficits with a high mortality rate. Early diagnosis and initiation of treatment are essential to kerb neurological deterioration. However, this is not always possible as 25% of cerebrospinal fluid samples produce false-negative results at first cytological examination. The identification of biomarkers that allow stratification of individuals according to risk for developing LM would be a major benefit. Proteomic-based approaches are now in increasing use for this purpose, and these are reviewed in this chapter with a focus on cerebrospinal fluid (CSF) analyses. The construction of a CSF proteome disease database would also facilitate analysis of other neurological disorders.

Multipronged functional proteomics approaches for global identification of altered cell signalling pathways in B-cell chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is a malignant B cell disorder characterized by its high heterogeneity. Although genomic alterations have been broadly reported, protein studies are still in their early stages. Herein, a 224-antibody microarray has been employed to study the intracellular signalling pathways in a cohort of 14 newly diagnosed B-CLL patients as a preliminary study for further investigations. Several protein profiles were differentially identified across the cytogenetic and molecular alterations presented in the samples (deletion 13q14 and 17p13.1, trisomy 12, and NOTCH1 mutations) by a combination of affinity and MS/MS proteomics approaches. Among others altered cell signalling pathways, PKC family members were identified as down-regulated in nearly 75% of the samples tested with the antibody arrays. This might explain the rapid progression of the disease when showing p53, Rb1, or NOTCH1 mutations due to PKC-proteins family plays a critical role favouring the slowly progressive indolent behaviour of CLL. Additionally, the antibody microarray results were validated by a LC-MS/MS quantification strategy and compared to a transcriptomic CLL database. In summary, this research displays the usefulness of proteomic strategies to globally evaluate the protein alterations in CLL cells and select the possible biomarkers to be further studied with larger sample sizes.

Novel reduced graphene oxide-glycol chitosan nanohybrid for the assembly of an amperometric enzyme biosensor for phenols.

A novel water-soluble graphene derivative was prepared from graphene oxide via a two-step modification approach. Graphene oxide was first functionalised with reactive epoxy groups by covalent modification with (3-glycidyloxypropyl)trimethoxysilane and further cross-linked with glycol chitosan. This graphene derivative was characterized using different microscopy and physicochemical methods and employed as a coating material for a glassy carbon electrode. The nanostructured surface was used as a support for the covalent immobilization of the enzyme laccase through cross-linking with glutaraldehyde. The enzyme electrode was tested for the amperometric detection of different phenolic compounds, which displayed excellent analytical behaviour toward catechol with a linear range of response from 200 nM to 15 µM, sensitivity of 93 mA M(-1) cm(-2), and low detection limit of 76 nM. The enzyme biosensor showed high stability when stored at 4 °C under dry conditions and was successfully employed to quantify the total phenolic compounds in commercial herbal tea samples.

Label-free electrochemical genosensor based on mesoporous silica thin film.

A novel label-free electrochemical strategy for nucleic acid detection was developed by using gold electrodes coated with mesoporous silica thin films as sensing interface. The biosensing approach relies on the covalent attachment of a capture DNA probe on the surface of the silica nanopores and further hybridization with its complementary target oligonucleotide sequence, causing a diffusion hindering of an Fe(CN)6 (3-/4-) electrochemical probe through the nanochannels of the mesoporous film. This DNA-mesoporous silica thin film-modified electrodes allowed sensitive (91.7 A/M) and rapid (45 min) detection of low nanomolar levels of synthetic target DNA (25 fmol) and were successfully employed to quantify the endogenous content of Escherichia coli 16S ribosomal RNA (rRNA) directly in raw bacterial lysate samples without isolation or purification steps. Moreover, the 1-month stability demonstrated by these biosensing devices enables their advanced preparation and storage, as desired for practical real-life applications. Graphical abstract Mesoporous silica thin films as scaffolds for the development of novel label-free electrochemical genosensors to perform selective, sensitive and rapid detection of target oligonucleotide sequences. Application towards E. coli determination.

Proteogenomics for the Comprehensive Analysis of Human Cellular and Serum Antibody Repertoires.

The vast repertoire of immunoglobulins produced by the immune system is a consequence of the huge amount of antigens to which we are exposed every day. The diversity of these immunoglobulins is due to different mechanisms (including VDJ recombination, somatic hypermutation, and antigen selection). Understanding how the immune system is capable of generating this diversity and which are the molecular bases of the composition of immunoglobulins are key challenges in the immunological field. During the last decades, several techniques have emerged as promising strategies to achieve these goals, but it is their combination which appears to be the fruitful solution for increasing the knowledge about human cellular and serum antibody repertoires.In this chapter, we address the diverse strategies focused on the analysis of immunoglobulin repertoires as well as the characterization of the genomic and peptide sequences. Moreover, the advantages of combining various -omics approaches are discussed through review different published studies, showing the benefits in clinical areas.

Integration of Proteomics and Transcriptomics Data Sets for the Analysis of a Lymphoma B-Cell Line in the Context of the Chromosome-Centric Human Proteome Project.

A comprehensive study of the molecular active landscape of human cells can be undertaken to integrate two different but complementary perspectives: transcriptomics, and proteomics. After the genome era, proteomics has emerged as a powerful tool to simultaneously identify and characterize the compendium of thousands of different proteins active in a cell. Thus, the Chromosome-centric Human Proteome Project (C-HPP) is promoting a full characterization of the human proteome combining high-throughput proteomics with the data derived from genome-wide expression profiling of protein-coding genes. Here we present a full proteomic profiling of a human lymphoma B-cell line (Ramos) performed using a nanoUPLC-LTQ-Orbitrap Velos proteomic platform, combined to an in-depth transcriptomic profiling of the same cell type. Data are available via ProteomeXchange with identifier PXD001933. Integration of the proteomic and transcriptomic data sets revealed a 94% overlap in the proteins identified by both -omics approaches. Moreover, functional enrichment analysis of the proteomic profiles showed an enrichment of several functions directly related to the biological and morphological characteristics of B-cells. In turn, about 30% of all protein-coding genes present in the whole human genome were identified as being expressed by the Ramos cells (stable average of 30% genes along all the chromosomes), revealing the size of the protein expression-set present in one specific human cell type. Additionally, the identification of missing proteins in our data sets has been reported, highlighting the power of the approach. Also, a comparison between neXtProt and UniProt database searches has been performed. In summary, our transcriptomic and proteomic experimental profiling provided a high coverage report of the expressed proteome from a human lymphoma B-cell type with a clear insight into the biological processes that characterized these cells. In this way, we demonstrated the usefulness of combining -omics for a comprehensive characterization of specific biological systems.

In Vitro Transcription/Translation System: A Versatile Tool in the Search for Missing Proteins.

Approximately 18% of all human genes purported to encode proteins have not been directly evidenced at the protein level, according to the validation criteria established by neXtProt, and are considered to be "missing" proteins. One of the goals of the Chromosome-Centric Human Proteome Project (C-HPP) is to identify as many of these missing proteins as possible in human samples using mass spectrometry-based methods. To further this goal, a consortium of C-HPP teams (chromosomes 5, 10, 16, and 19) has joined forces to devise new strategies to identify missing proteins by use of a cell-free in vitro transcription/translation system (IVTT). The proposed strategy employs LC-MS/MS data-dependent acquisition (DDA) and targeted selective reaction monitoring (SRM) methods to scrutinize low-complexity samples derived from IVTT. The optimized assays are then applied to identify missing proteins in human cells and tissues. We describe the approach and show proof-of-concept results for development of LC-SRM assays for identification of 18 missing proteins. We believe that the IVTT system, when coupled with downstream mass spectrometric identification, can be applied to identify proteins that have eluded more traditional methods of detection.

Quest for Missing Proteins: Update 2015 on Chromosome-Centric Human Proteome Project.

This paper summarizes the recent activities of the Chromosome-Centric Human Proteome Project (C-HPP) consortium, which develops new technologies to identify yet-to-be annotated proteins (termed "missing proteins") in biological samples that lack sufficient experimental evidence at the protein level for confident protein identification. The C-HPP also aims to identify new protein forms that may be caused by genetic variability, post-translational modifications, and alternative splicing. Proteogenomic data integration forms the basis of the C-HPP's activities; therefore, we have summarized some of the key approaches and their roles in the project. We present new analytical technologies that improve the chemical space and lower detection limits coupled to bioinformatics tools and some publicly available resources that can be used to improve data analysis or support the development of analytical assays. Most of this paper's content has been compiled from posters, slides, and discussions presented in the series of C-HPP workshops held during 2014. All data (posters, presentations) used are available at the C-HPP Wiki (http://c-hpp.webhosting.rug.nl/) and in the Supporting Information.

Toward the design of smart delivery systems controlled by integrated enzyme-based biocomputing ensembles.

We report herein the design of a smart delivery system in which cargo delivery from capped mesoporous silica (MS) nanoparticles is controlled by an integrated enzyme-based "control unit". The system consists of Janus-type nanoparticles having opposing Au and MS faces, functionalized with a pH-responsive ß-cyclodextrin-based supramolecular nanovalve on the MS surface and two effectors, glucose oxidase and esterase, immobilized on the Au face. The nanodevice behaves as an enzymatic logical OR operator which is selectively fueled by the presence of D-glucose and ethyl butyrate.

Biomarkers in Ovarian Cancer: Towards Personalized Medicine.

Ovarian cancer is one of the deadliest cancers in women. The lack of specific symptoms, especially at the initial stages of disease development, together with the malignancy heterogeneity, lower the life expectancy of patients. Aiming to improve survival rates, diagnostic and prognostic biomarkers are increasingly employed in clinics, providing gynecologists and oncologists with new tools to guide their treatment decisions. Despite the vast number of investigations, there is still an urgent need to discover more ovarian cancer subtype-specific markers which could further improve patient classification. To this end, high-throughput screening technologies, like mass spectrometry, are applied to deepen the tumoral cellular landscape and describe the malignant phenotypes. As for disease treatment, new targeted therapies, such as those based on PARP inhibitors, have shown great efficacy in destroying the tumoral cells. Likewise, drug-nanocarrier systems targeting the tumoral cells have exhibited promising results. In this narrative review, we summarize the latest achievements in the pursuit of biomarkers for ovarian cancer and recent anti-tumoral therapies.