The first book to offer a blueprint for overcoming the challenges to successfully quantifying biomarkers in living organisms The demand among scientists and clinicians for targeted quantitation experiments has experienced explosive growth in recent years. While there are a few books dedicated to bioanalysis and biomarkers in general, until now there were none devoted exclusively to addressing critical issues surrounding this area of intense research. Target Biomarker Quantitation by LC-MS provides a detailed blueprint for quantifying biomarkers in biological systems. It uses numerous real-world cases to exemplify key concepts, all of which were carefully selected and presented so as to allow the concepts they embody to be easily expanded to future applications, including new biomarker development. Target Biomarker Quantitation by LC-MS primarily focuses on the assay establishment for biomarker quantitation—a critical issue rarely treated in depth. It offers comprehensive coverage of three core areas of biomarker assay establishment: the relationship between the measured biomarkers and their intended usage; contemporary regulatory requirements for biomarker assays (a thorough understanding of which is essential to producing a successful and defendable submission); and the technical challenges of analyzing biomarkers produced inside a living organism or cell. Covers the theory of and applications for state-of-the-art mass spectrometry and chromatography and their applications in biomarker analysis Features real-life examples illustrating the challenges involved in target biomarker quantitation and the innovative approaches which have been used to overcome those challenges Addresses potential obstacles to obtain effective biomarker level and data interpretation, such as specificity establishment and sample collection Outlines a tiered approach and fit-for-purpose assay protocol for target biomarker quantitation Highlights the current state of the biomarker regulatory environment and protocol standards Target Biomarker Quantitation by LC-MS is a valuable resource for bioanalytical scientists, drug metabolism and pharmacokinetics scientists, clinical scientists, analytical chemists, and others for whom biomarker quantitation is an important tool of the trade. It also functions as an excellent text for graduate courses in pharmaceutical, biochemistry and chemistry.

This book is oriented towards post-graduates and researchers with interest in proteomics and its applications in clinical biomarker discovery pipeline. Biomarker discovery has long been the research focus of many life scientists globally. However, the pipeline starting from discovery to validation to regulation as a diagnostic or therapeutic molecule follows a complex trajectory. This book aims to provide an in-depth synopsis on each of these developmental phases attendant to biomarker “life cycle” with emphasis on the emerging and significant role of proteomics. The book begins with a perspective on the role of biorepositories and need for biobanking practices in the developing world. The next chapter focuses on disease heterogeneity in context to geographical bias towards susceptibility to the disease and the role of multi-omics techniques to devise disruptive innovations towards biomarker discovery. Chapter 3 focuses on various omics-based platforms that are currently being used for biomarker discovery, their principles and workflow. Mass spectrometry is emerging as a powerful technology for discovery based studies and targeted validation. Chapter 4 aims at providing a glimpse of the basic workflow and considerations in mass spectrometry based studies. Rapid and aptly targeted research funding has often been deemed as one of the decisive factors enabling excellent science and path breaking innovations. With the need for sophistication required in multi-omics research, Chapter 5 focuses on innovative funding strategies such as crowdfunding and Angel philanthropy. Chapter 6 provides the latest advances in education innovation, the premise and reality of bioeconomy especially in a specific context of the developing world, not to mention the new concept of “social innovation” to link biomarkers with socially responsible and sustainable applications. Chapter 7, in ways similar to biomarkers, discusses the biosimilars as a field that has received much focus and prominence recently due to their immense potential in clinical and pharmaceutical innovation literatures. The broader goal post-biomarker discovery is to translate their use in clinics. However, the road from bench-to-bed side is arduous and complex that is subject to oversight from various national and international regulatory bodies. Chapter 8 underscores these regulatory science considerations and provides a concise overview on intellectual property rights in biomarker discovery. Thus, this book contributed by eminent biomarker scientists, clinicians, translational researchers and social scientists holistically covers the various facets of the biomarker discovery journey from “cell to society” in developing world. The lessons learned and highlighted here are of interest to the life sciences community in a global and interdependent world.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has seen enormous growth in the clinical space in the past decade. In the United States, majority of the clinical reference laboratories use LC-MS/MS for the steroid assays due to its ability to offer analytical specificity, sensitivity, and high-throughput which are limitations of immunoassays. This technique has also shown its potential in biomarker studies, deciphering the biochemical events, and its application to answer clinically challenging questions has helped to progress in the direction of precision medicine. In this work, we have exploited the LC-MS/MS characteristics of specificity and sensitivity to develop the first validated method for the quantitation of dihydrotestosterone (DHT) and its metabolites in various biomatrices such as mouse serum, mouse prostate tissue and supernatant of cell culture media. Chemical derivatization was used to improve the detection limits of the analytes. The calibration range for DHT and its metabolites was 0.0500 - 50.0 ng/mL. The principles of method development, and validation according to FDA-ICH guidelines are depicted in this quantitative bioassay. In the second half of this publication, we showed the synergistic capabilities of high-resolution mass spectrometry in combination with the new bioinformatic tools to perform a multiomic analysis of exosome cargo in which the proteins and lipids were profiled, semi-quantitated, statistically modeled, and differentially analyzed to predict the molecular mechanisms through the protein-protein interaction networks. We showed for the first time the sample preparation steps which enabled the simultaneous extraction of proteins and lipids from same exosome sample. We used two sets of cell lines of human T-cells i.e., control and infected with HIV-1 as a model for this study. A number of open source bioinformatic tools were used for the identification, semi quantitation, functional enrichment, pathway analysis, and protein interaction networks to predict the possible molecular mechanisms.

The first book to offer a blueprint for overcoming the challenges to successfully quantifying biomarkers in living organisms The demand among scientists and clinicians for targeted quantitation experiments has experienced explosive growth in recent years. While there are a few books dedicated to bioanalysis and biomarkers in general, until now there were none devoted exclusively to addressing critical issues surrounding this area of intense research. Target Biomarker Quantitation by LC-MS provides a detailed blueprint for quantifying biomarkers in biological systems. It uses numerous real-world cases to exemplify key concepts, all of which were carefully selected and presented so as to allow the concepts they embody to be easily expanded to future applications, including new biomarker development. Target Biomarker Quantitation by LC-MS primarily focuses on the assay establishment for biomarker quantitation—a critical issue rarely treated in depth. It offers comprehensive coverage of three core areas of biomarker assay establishment: the relationship between the measured biomarkers and their intended usage; contemporary regulatory requirements for biomarker assays (a thorough understanding of which is essential to producing a successful and defendable submission); and the technical challenges of analyzing biomarkers produced inside a living organism or cell. Covers the theory of and applications for state-of-the-art mass spectrometry and chromatography and their applications in biomarker analysis Features real-life examples illustrating the challenges involved in target biomarker quantitation and the innovative approaches which have been used to overcome those challenges Addresses potential obstacles to obtain effective biomarker level and data interpretation, such as specificity establishment and sample collection Outlines a tiered approach and fit-for-purpose assay protocol for target biomarker quantitation Highlights the current state of the biomarker regulatory environment and protocol standards Target Biomarker Quantitation by LC-MS is a valuable resource for bioanalytical scientists, drug metabolism and pharmacokinetics scientists, clinical scientists, analytical chemists, and others for whom biomarker quantitation is an important tool of the trade. It also functions as an excellent text for graduate courses in pharmaceutical, biochemistry and chemistry.

Proteomic and Metabolomic Approaches to Biomarker Discovery, Second Edition covers techniques from both proteomics and metabolomics and includes all steps involved in biomarker discovery, from study design to study execution. The book describes methods and presents a standard operating procedure for sample selection, preparation and storage, as well as data analysis and modeling. This new standard effectively eliminates the differing methodologies used in studies and creates a unified approach. Readers will learn the advantages and disadvantages of the various techniques discussed, as well as potential difficulties inherent to all steps in the biomarker discovery process. This second edition has been fully updated and revised to address recent advances in MS and NMR instrumentation, high-field NMR, proteomics and metabolomics for biomarker validation, clinical assays of biomarkers and clinical MS and NMR, identifying microRNAs and autoantibodies as biomarkers, MRM-MS assay development, top-down MS, glycosylation-based serum biomarkers, cell surface proteins in biomarker discovery, lipodomics for cancer biomarker discovery, and strategies to design studies to identify predictive biomarkers in cancer research. Addresses the full range of proteomic and metabolomic methods and technologies used for biomarker discovery and validation Covers all steps involved in biomarker discovery, from study design to study execution Serves as a vital resource for biochemists, biologists, analytical chemists, bioanalytical chemists, clinical and medical technicians, researchers in pharmaceuticals and graduate students

Consolidates the information LC-MS bioanalytical scientists need to analyze small molecules and macromolecules The field of bioanalysis has advanced rapidly, propelled by new approaches for developing bioanalytical methods, new liquid chromatographic (LC) techniques, and new mass spectrometric (MS) instruments. Moreover, there are a host of guidelines and regulations designed to ensure the quality of bioanalytical results. Presenting the best practices, experimental protocols, and the latest understanding of regulations, this book offers a comprehensive review of LC-MS bioanalysis of small molecules and macromolecules. It not only addresses the needs of bioanalytical scientists working on routine projects, but also explores advanced and emerging technologies such as high-resolution mass spectrometry and dried blood spot microsampling. Handbook of LC-MS Bioanalysis features contributions from an international team of leading bioanalytical scientists. Their contributions reflect a review of the latest findings, practices, and regulations as well as their own firsthand analytical laboratory experience. The book thoroughly examines: Fundamentals of LC-MS bioanalysis in drug discovery, drug development, and therapeutic drug monitoring The current understanding of regulations governing LC-MS bioanalysis Best practices and detailed technical instructions for LC-MS bioanalysis method development, validation, and stability assessment of analyte(s) of interest Experimental guidelines and protocols for quantitative LC-MS bioanalysis of challenging molecules, including pro-drugs, acyl glucuronides, N-oxides, reactive compounds, and photosensitive and autooxidative compounds With its focus on current bioanalytical practice, Handbook of LC-MS Bioanalysis enables bioanalytical scientists to develop and validate robust LC-MS assay methods, all in compliance with current regulations and standards.