Biomarker Discovery by Novel Sensors Based on Nanoproteomics Approaches

During the last years, proteomics has facilitated biomarker discovery by coupling high-throughput techniques with novel nanosensors. In the present review, we focus on the study of label-based and label-free detection systems, as well as nanotechnology approaches, indicating their advantages and applications in biomarker discovery. In addition, several disease biomarkers are shown in order to display the clinical importance of the improvement of sensitivity and selectivity by using nanoproteomics approaches as novel sensors

Nanotechnology in proteomics: Current status, promises and challenges

In genomics, the ability to amplify rare transcripts has enabled rapid advances in the understanding of gene expression patterns in human disease. The inability to increase the copy number and to detect the signal of rare proteins as unique species in biological samples has hindered the ability of proteomics to dissect human disease with the same complexity as genomic analyses. Advances in nanotechnology have begun to allow researchers to identify low-abundance proteins in samples through techniques that rely upon both nanoparticles and nanoscale devices. This review describes some of the physical and chemical principles underlying nanomaterials and devices and outlines how they can be used in proteomics; developments which are establishing nanoproteomics as a new field. Nanoproteomics will provide the platform for the discovery of next generation biomarkers. The most promising candidates for nanoproteomics, namely carbon nanotubes and nanowires, quantum dots and nanoscopic gold particles, offer several advantages such as high sensitivity, real-time measurements and improved reproducibility.

Nanogenomics and Nanoproteomics Enabling Personalized, Predictive and Preventive Medicine

Since the discovery of the nucleic acid, molecular biology has made tremendous progresses, achieving a lot of results. Despite this, there is still a gap between the classical and traditional medical approach and the molecular world. Inspired by the incredible wealth of data generated by the "omics"-driven techniques and the “high-trouhgput technologies” (HTTs), I have tried to develop a protocol that could reduce the actually extant barrier between the phenomenological medicine and the molecular medicine, facilitating a translational shift from the lab to the patient bedside. I also felt the urgent need to integrate the most important omics sciences, that is to say genomics and proteomics. Nucleic Acid Programmable Protein Arrays (NAPPA) can do this, by utilizing a complex mammalian cell free expression system to produce proteins in situ. In alternative to fluorescent-labeled approaches a new label free method, emerging from the combined utilization of three independent and complementary nanobiotechnological approaches, appears capable to analyze gene and protein function, gene-protein, gene-drug, protein-protein and protein-drug interactions in studies promising for personalized medicine. Quartz Micro Circuit nanogravimetry (QCM), based on frequency and dissipation factor, mass spectrometry (MS) and anodic porous alumina (APA) overcomes indeed the limits of correlated fluorescence detection plagued by the background still present after extensive washes. Work is in progress to further optimize this approach a homogeneous and well defined bacterial cell free expression system able to realize the ambitious objective to quantify the regulatory gene and protein networks in humans. Implications for personalized medicine of the above label free protein array using different test genes and proteins are reported in this PhD thesis

Microarrays as Platform for Multiplex Assays in Biomarker and Drug Discovery

Despite the tremendous advances in the understanding of the molecular mechanisms and the complexity of the diseases is one of the present challenges for the scientific community; then, novel strategies are required to be designed and developed for effective strategies for early diagnosis and treatment. As many cellular alterations are observed at protein level, high-throughput assays are dramatically needed for biomarker discovery. Herein, we describe advantages and limitations of protein microarrays, as proteomics strategy useful for multiplex and high-throughput protein characterization in clinical samples. Finally, a few examples are discussed; mostly of them related to currently disease biomarkers already identified in proximal fluids by protein arrays are discussed

NAPPA as a Real New Method for Protein Microarray Generation

abstract: Nucleic Acid Programmable Protein Arrays (NAPPA) have emerged as a powerful and innovative technology for the screening of biomarkers and the study of protein-protein interactions, among others possible applications. The principal advantages are the high specificity and sensitivity that this platform offers. Moreover, compared to conventional protein microarrays, NAPPA technology avoids the necessity of protein purification, which is expensive and time-consuming, by substituting expression in situ with an in vitro transcription/translation kit. In summary, NAPPA arrays have been broadly employed in different studies improving knowledge about diseases and responses to treatments. Here, we review the principal advances and applications performed using this platform during the last years

Biomarkers of inflammatory arthritis and proteomics

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Determination of Protein-Protein Interaction for Cancer Control via Mass Spectrometry and Nanoconductimetry of NAPPA SNAP Arrays: An Overview

Background: Protein-protein interactions play a major role in Cancer Control and their detailed understanding by Label-Free Nanotechnology is essential especially within the framework of a personalized medicine-based approach. Material and Methods: We implemented an array of label-free nanobiotechnologies, including the Quartz Crystal Microbalance with Dissipation factor monitoring (QCM_D). We used it for the conductometric monitoring of an antiblastic (temozolomide) interacting with genes and proteins, such as MLH1, that represents a biomarker of the rate survival of patients suffering from brain tumors, outcome of chemotherapy and resistance to drug itself. We coupled the Nucleic Acid Programmable Protein Arrays (NAPPA) and the cell-free protein array with the quartz crystal microbalance technology. In another proof of principle, we coupled the NAPPA with the SNAP tag E. colicell-free expression system. The goal is to analyze the protein-protein interaction using Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Bruker Ultraflex and \u201cProtein synthesis Using Recombinant Elements\u201d (PURE) system, thus avoiding the \u201cblack box\u201d nature of the cell extract. The E. coliin vitro transcription/translation system (IVTT) in respect to the reticulocyte lysate (RRL) or human lysate (HL) is totally characterized and represents an advantage for the subsequent mass spectrometry (MS) analysis. An R Script for Mass Spectrometry Data Preprocessing before Data Mining (SpADS) provides the user with peak recognition and amplitude independent subtraction functions. The MS samples are obtained from SNAP-NAPPA spots and printed on gold coated glass slides in higher density, in order to obtain an amount of protein appropriate for MS analysis. Conclusion: We developed a coherent approach that overcome the drawbacks and pitfalls of the traditional laborious and time-consuming labeled and fluorescence-based experimental procedures. This, taken together with the unique properties of proteins obtained with Langmuir-Blodgett (LB)-based crystallography that can enable new strategies for drug design separately reported, defines our approach to cancer control

A flavour of omics approaches for the detection of food fraud

Food fraud has been identified as an increasing problem on a global scale with wide-ranging economic, social, health and environmental impacts. Omics and their related techniques, approaches, and bioanalytical platforms incorporate a significant number of scientific areas which have the potential to be applied to and significantly reduce food fraud and its negative impacts. In this overview we consider a selected number of very recent studies where omics techniques were applied to detect food authenticity and could be implemented to ensure food integrity. We postulate that significant reductions in food fraud, with the assistance of omics technologies and other approaches, will result in less food waste, decreases in energy use as well as greenhouse gas emissions, and as a direct consequence of this, increases in quality, productivity, yields, and the ability of food systems to be more resilient and able to withstand future food shocks

Nanotechnology and Its Applications in Medical Diagnosis

ABSTRACT Nanobiotechnology and its applications in life sciences particularly in molecular diagnostics are termed as Nanomolecular diagnostics, which offers new options for clinical diagnostic procedures. Molecular diagnostics is an essential part of the development of personalized medicine, which features point-ofcare performance of diagnostic procedure. Nanobiotechnology incorporated along with molecular diagnostics improves the clinical diagnosis tremendously. In this type of diagnosis, 'Nanoscale probes' are mostly suitable for detailed analysis of receptors, pores and other components of living cells that exist in nanoscale dimension. For combined diagnosis and therapeutics, Nanodevice can be implanted as a preventive and prophylactic measure in early disease diagnosis. Advantages of applying nanotechnology to molecular diagnostics are that only small amounts of sample material are needed and that diagnostic tests that use nanoscale particles as tags or labels are faster and more sensitive 1

Deciphering biomarkers for leptomeningeal metastasis in malignant hemopathies (Lymphoma/Leukemia) patients by comprehensive multipronged proteomics characterization of cerebrospinal fluid

In the present work, leptomeningeal disease, a very destructive form of systemic cancer, was characterized from several proteomics points of view. This pathology involves the invasion of the leptomeninges by malignant tumor cells. The tumor spreads to the central nervous system through the cerebrospinal fluid (CSF) and has a very grim prognosis; the average life expectancy of patients who suffer it does not exceed 3 months. The early diagnosis of leptomeningeal disease is a challenge because, in most of the cases, it is an asymptomatic pathology. When the symptoms are clear, the disease is already in the very advanced stages and life expectancy is low. Consequently, there is a pressing need to determine useful CSF proteins to help in the diagnosis and/or prognosis of this disease. For this purpose, a systematic and exhaustive proteomics characterization of CSF by multipronged proteomics approaches was performed to determine different protein profiles as potential biomarkers. Proteins such as PTPRC, SERPINC1, sCD44, sCD14, ANPEP, SPP1, FCGR1A, C9, sCD19, and sCD34, among others, and their functional analysis, reveals that most of them are linked to the pathology and are not detected on normal CSF. Finally, a panel of biomarkers was verified by a prediction model for leptomeningeal disease, showing new insights into the research for potential biomarkers that are easy to translate into the clinic for the diagnosis of this devastating disease.We gratefully acknowledge financial support from the Spanish Health Institute, Carlos III (ISCIII), for the grants: FIS PI14/01538, FIS PI17/01930 and CB16/12/00400. We also acknowledge Fondos FEDER (EU) and Junta Castilla-León (COVID-19 grant COV20EDU/00187). The Proteomics Unit belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019/0023 of the PE I + D + I2017-2020, funded by ISCIII and FEDER—Norma Galicia is supported by the CONACYT Program. P. Juanes-Velasco is supported by JCYL PhD Program “Nos Impulsa-JCYL” and scholarship JCYLEDU/601/2020