Toward a Consensus on Applying Quantitative Liquid Chromatography-Tandem Mass Spectrometry Proteomics in Translational Pharmacology Research: A White Paper.

Quantitative translation of information on drug absorption, disposition, receptor engagement, and drug-drug interactions from bench to bedside requires models informed by physiological parameters that link in vitro studies to in vivo outcomes. To predict in vivo outcomes, biochemical data from experimental systems are routinely scaled using protein quantity in these systems and relevant tissues. Although several laboratories have generated useful quantitative proteomic data using state-of-the-art mass spectrometry, no harmonized guidelines exit for sample analysis and data integration to in vivo translation practices. To address this gap, a workshop was held on September 27 and 28, 2018, in Cambridge, MA, with 100 experts attending from academia, the pharmaceutical industry, and regulators. Various aspects of quantitative proteomics and its applications in translational pharmacology were debated. A summary of discussions and best practices identified by this expert panel are presented in this "White Paper" alongside unresolved issues that were outlined for future debates.

Accelerating Biomarker Discovery Through Electronic Health Records, Automated Biobanking, and Proteomics.

BACKGROUND: Circulating biomarkers can facilitate diagnosis and risk stratification for complex conditions such as heart failure (HF). Newer molecular platforms can accelerate biomarker discovery, but they require significant resources for data and sample acquisition. OBJECTIVES: The purpose of this study was to test a pragmatic biomarker discovery strategy integrating automated clinical biobanking with proteomics. METHODS: Using the electronic health record, the authors identified patients with and without HF, retrieved their discarded plasma samples, and screened these specimens using a DNA aptamer-based proteomic platform (1,129 proteins). Candidate biomarkers were validated in 3 different prospective cohorts. RESULTS: In an automated manner, plasma samples from 1,315 patients (31% with HF) were collected. Proteomic analysis of a 96-patient subset identified 9 candidate biomarkers (p < 4.42 × 10-5). Two proteins, angiopoietin-2 and thrombospondin-2, were associated with HF in 3 separate validation cohorts. In an emergency department-based registry of 852 dyspneic patients, the 2 biomarkers improved discrimination of acute HF compared with a clinical score (p < 0.0001) or clinical score plus B-type natriuretic peptide (p = 0.02). In a community-based cohort (n = 768), both biomarkers predicted incident HF independent of traditional risk factors and N-terminal pro-B-type natriuretic peptide (hazard ratio per SD increment: 1.35 [95% confidence interval: 1.14 to 1.61; p = 0.0007] for angiopoietin-2, and 1.37 [95% confidence interval: 1.06 to 1.79; p = 0.02] for thrombospondin-2). Among 30 advanced HF patients, concentrations of both biomarkers declined (80% to 84%) following cardiac transplant (p < 0.001 for both). CONCLUSIONS: A novel strategy integrating electronic health records, discarded clinical specimens, and proteomics identified 2 biomarkers that robustly predict HF across diverse clinical settings. This approach could accelerate biomarker discovery for many diseases.

Proceedings of the EuBIC developer's meeting 2018.

The inaugural European Bioinformatics Community (EuBIC) developer's meeting was held from January 9th to January 12th 2018 in Ghent, Belgium. While the meeting kicked off with an interactive keynote session featuring four internationally renowned experts in the field of computational proteomics, its primary focus were the hands-on hackathon sessions which featured six community-proposed projects revolving around three major topics: Here, we present an overview of the scientific program of the EuBIC developer's meeting and provide a starting point for follow-up on the covered projects.

Application of Proteomic Techniques for Improved Stratification and Treatment of Schizophrenia Patients.

For major psychiatric disorders such as schizophrenia, there have been shortcomings in the translation of scientific findings into new treatments and this has led to diminished interest for large pharmaceutical companies. This chapter describes how incorporation of proteomic approaches into the clinical pipeline can lead to identification and implementation of biomarker tests for improved patient characterization, prediction of treatment response and monitoring treatment effects to help revitalize efforts in this important area. In addition, the construction of specific biomarker tests for disease prediction should smooth the progress of early intervention strategies which, in turn, may help to slow disease onset or progression. Finally, the development of purpose-built biomarker tests using lab-on-a-chip platforms with smartphone readouts will help to shift the diagnosis and treatment of this major psychiatric disorder into point-of-care settings for increased effectiveness and improved patient outcomes.

A multicentric study to evaluate the use of relative retention times in targeted proteomics.

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. BIOLOGICAL SIGNIFICANCE: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups.

A decade of Central and Eastern European Proteomic Conference (CEEPC): Credibility, cohesion and vision for the next decade.

The Central and Eastern European Proteomic Conference (CEEPC), has reached a special milestone as it celebrates its 10th anniversary. Today, an expansive network of proteomics in Central and Eastern Europe stands established to facilitate scientific interactions and collaborations in and around Central and Eastern Europe, as well as with international research institutions worldwide. Currently, when many conferences are struggling to attract participants, CEEPC is thriving in its status and stature as well as expanding by attracting newer member countries. CEEPC's success is driven by mutual respect between scientists sharing interest in proteomics and its applications in multidisciplinary research areas related to biological systems. This effort when interwoven with exciting ambience steeped with culture, and tradition is also a reason why participants enjoy it. CEEPC's careful balance between excellence and cohesion holds the key to its success. It is evident that CEEPC is ready for the next decade of excitement and expectations of multifaceted proteomics in Central and Eastern Europe. Additionally, in the era of emerging personalized medicine where treatment selection for each patient is becoming individualized, CEEPC and proteomics is expected to play a significant role moving forward for the benefit of mankind.

Human Proteome Project Mass Spectrometry Data Interpretation Guidelines 2.1.

Every data-rich community research effort requires a clear plan for ensuring the quality of the data interpretation and comparability of analyses. To address this need within the Human Proteome Project (HPP) of the Human Proteome Organization (HUPO), we have developed through broad consultation a set of mass spectrometry data interpretation guidelines that should be applied to all HPP data contributions. For submission of manuscripts reporting HPP protein identification results, the guidelines are presented as a one-page checklist containing 15 essential points followed by two pages of expanded description of each. Here we present an overview of the guidelines and provide an in-depth description of each of the 15 elements to facilitate understanding of the intentions and rationale behind the guidelines, for both authors and reviewers. Broadly, these guidelines provide specific directions regarding how HPP data are to be submitted to mass spectrometry data repositories, how error analysis should be presented, and how detection of novel proteins should be supported with additional confirmatory evidence. These guidelines, developed by the HPP community, are presented to the broader scientific community for further discussion.

Constructing failure in big biology: The socio-technical anatomy of Japan's Protein 3000 Project.

This study focuses on the 5-year Protein 3000 Project launched in 2002, the largest biological project in Japan. The project aimed to overcome Japan's alleged failure to contribute fully to the Human Genome Project, by determining 3000 protein structures, 30 percent of the global target. Despite its achievement of this goal, the project was fiercely criticized in various sectors of society and was often branded an awkward failure. This article tries to solve the mystery of why such failure discourse was prevalent. Three explanatory factors are offered: first, because some goals were excluded during project development, there was a dynamic of failed expectations; second, structural genomics, while promoting collaboration with the international community, became an 'anti-boundary object', only the absence of which bound heterogeneous domestic actors; third, there developed an urgent sense of international competition in order to obtain patents on such structural information.

Key players in neurodegenerative disorders in focus-New insights into the proteomic profile of Alzheimer's disease, schizophrenia, ALS, and multiple sclerosis-24th HUPO BPP Workshop: September 29, 2015, Vancouver, Canada.

The HUPO Brain Proteome Project (HUPO BPP) held its 24th workshop in Vancouver, Canada, September 29, 2015. The focus of the autumn workshop was on new insights into the proteomic profile of Alzheimer's disease, schizophrenia, ALS and multiple sclerosis.

The intra-tumor heterogeneity of cell signaling factors in breast cancer: p4E-BP1 and peIF4E are diffusely expressed and are real potential targets

Breast cancers and most malignant tumors are composed of heterogeneous tumor cells both at genetic and morphological levels; intra-tumor heterogeneity can be one underlying cause of therapeutic resistance. Classical studies have focused on analyses of the relationship between primary tumors and metastatic dissemination, and on subclone evolution. However, it should be noted that tumor heterogeneity at the level of protein expression (proteomics) has not been yet studied in depth. The differences in protein expression also can play an important role in elucidating the relationship between intra-tumor heterogeneity and resistance to systemic therapy. In fact, in human tumors there is not always a homogeneous expression of many of the crucial factors involved in cell signaling, such as pMAPK, pAKt, pMTOR, even with constitutive oncogenic alterations upstream, such as HER2, PI3 K. Conversely, two of these factors, peIF4E and p4E-BP1, which are downstream, and control protein translation, show a diffuse and strong protein expression. In summary, most of cell signaling factors show a heterogeneous expression, regardless of oncogenic alterations. Tissue heterogeneity could be driven by local factors, including hypoxia. The fact that the phosphorylation of crucial proteins such as 4E-BP1 and eIF4E is observed homogeneously throughout most tumors and are druggable opens the chance to get real potential targets in cancer therapy (AU)

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PRIME-XS, a European infrastructure for proteomics.

The PRIME-XS consortium is a pan-European infrastructure for proteomics. As a prologue to this special issue of Molecular & Cellular Proteomics on the research activities of the PRIME-XS consortium, we, as the guest editors of this issue, provide an overview of the structure and activities of this consortium, which is funded by the European Union's 7th Framework Programme for Research and Technological Development.

The strategy, organization, and progress of the HUPO Human Proteome Project.

The Human Proteome Project is a major, comprehensive initiative of the Human Proteome Organization. This global collaborative effort aims to identify and characterize at least one protein product and many PTM, SAP, and splice variant isoforms from the 20,300 human protein-coding genes. The deliverables are an extensive parts list and an array of technology platforms, reagents, spectral libraries, and linked knowledge bases that advance the field and facilitate the use of proteomics by a much wider community of life scientists. Such enablement will help address the Grand Challenge of using proteomics to bridge major gaps between evidence of genomic variation and diverse phenotypes. BIOLOGICAL SIGNIFICANCE: The HUPO Human Proteome Project (HPP) has made an outstanding launch, including a special issue of the Journal of Proteome Research on the Chromosome-centric HPP with a total of 48 articles. This article is part of a Special Issue: Can Proteomics Fill the Gap Between Genomics and Phenotypes?

Commercial considerations for immunoproteomics.

The underlying drivers of scientific processes have been rapidly evolving, but the ever-present need for research funding is typically foremost amongst these. Successful laboratories are embracing this reality by making certain that their projects have commercial value right from the beginning of the project conception. Which factors to be considered for commercial success need to be well thought out and incorporated into a project plan with similar levels of detail as would be the technical elements. Specific examples of commercial outcomes in the field of Immunoproteomics are exemplified in this discussion.

La proteómica y la metabolómica: los mecanismos de la enfermedad cardiovascular y el descubrimiento de biomarcadores / Proteomics and metabolomics for mechanistic insights and biomarker discovery in cardiovascular disease

En la última década, la proteómica y la metabolómica han realizado aportaciones sustanciales al conocimiento de las enfermedades cardiovasculares. La evaluación no sesgada de los procesos fisiopatológicos sin partir de presunciones establecidas a priori complementa otras técnicas de biología molecular que se emplean en la actualidad con un enfoque reduccionista. En la presente revisión, se resaltan algunos de los métodos de las ciencias «ómicas» que se emplean para evaluar los cambios de las proteínas y los metabolitos en la enfermedad cardiovascular. Es muy infrecuente que una función biológica específica discreta se atribuya a una sola molécula; es más habitual que se lleve a cabo con la aportación combinada de muchas proteínas. A diferencia del enfoque reduccionista, en el que se estudia individualmente las moléculas, las plataformas «ómicas» permiten el estudio de interacciones más complejas en sistemas biológicos. La combinación de la proteómica y la metabolómica para cuantificar los cambios de los metabolitos y sus correspondientes enzimas hará avanzar nuestro conocimiento de los mecanismos fisiopatológicos y facilitará la identificación de nuevos biomarcadores de enfermedad cardiovascular (AU)

In the last decade, proteomics and metabolomics have contributed substantially to our understanding of cardiovascular diseases. The unbiased assessment of pathophysiological processes without a priori assumptions complements other molecular biology techniques that are currently used in a reductionist approach. In this review, we highlight some of the «omics» methods used to assess protein and metabolite changes in cardiovascular disease. A discrete biological function is very rarely attributed to a single molecule; more often it is the combined input of many proteins. In contrast to the reductionist approach, in which molecules are studied individually, «omics» platforms allow the study of more complex interactions in biological systems. Combining proteomics and metabolomics to quantify changes in metabolites and their corresponding enzymes will advance our understanding of pathophysiological mechanisms and aid the identification of novel biomarkers for cardiovascular disease (AU)

Improving international research with clinical specimens: 5 achievable objectives.

Our increased interest in translational research has created a large demand for blood, tissue, and other clinical samples, which find use in a broad variety of research including genomics, proteomics, and metabolomics. Hundreds of millions of dollars have been invested internationally on the collection, storage, and distribution of samples. Nevertheless, many researchers complain in frustration about their inability to obtain relevant and/or useful samples for their research. Lack of access to samples, poor condition of samples, and unavailability of appropriate control samples have slowed our progress in the study of diseases and biomarkers. In this perspective, I focus on five major challenges that thwart clinical sample use for translational research and propose near term objectives to address them. They include: (1) defining our biobanking needs; (2) increasing the use of and access to standard operating procedures; (3) mapping interobserver differences for use in normalizing diagnoses; (4) identifying natural internal protein controls; and (5) redefining the clinical sample paradigm by building partnerships with the public. In each case, I believe that we have the tools at hand required to achieve the objective within 5 years. Potential paths to achieve these objectives are explored. However we solve these problems, the future of proteomics depends on access to high quality clinical samples, collected under standardized conditions, accurately annotated and shared under conditions that promote the research we need to do.

TP Atlas: integration and dissemination of advances in Targeted Proteins Research Program (TPRP)-structural biology project phase II in Japan.

The Targeted Proteins Research Program (TPRP) promoted by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan is the phase II of structural biology project (2007-2011) following the Protein 3000 Project (2002-2006) in Japan. While the phase I Protein 3000 Project put partial emphasis on the construction and maintenance of pipelines for structural analyses, the TPRP is dedicated to revealing the structures and functions of the targeted proteins that have great importance in both basic research and industrial applications. To pursue this objective, 35 Targeted Proteins (TP) Projects selected in the three areas of fundamental biology, medicine and pharmacology, and food and environment are tightly collaborated with 10 Advanced Technology (AT) Projects in the four fields of protein production, structural analyses, chemical library and screening, and information platform. Here, the outlines and achievements of the 35 TP Projects are summarized in the system named TP Atlas. Progress in the diversified areas is described in the modules of Graphical Summary, General Summary, Tabular Summary, and Structure Gallery of the TP Atlas in the standard and unified format. Advances in TP Projects owing to novel technologies stemmed from AT Projects and collaborative research among TP Projects are illustrated as a hallmark of the Program. The TP Atlas can be accessed at http://net.genes.nig.ac.jp/tpatlas/index_e.html .