INTRODUCTION: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. AREAS COVERED: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. EXPERT COMMENTARY: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine.

Analytical Chemistry Department Faculty of Pharmacy Iuliu Hatieganu University of Medicine and Pharmacy Cluj Napoca Romania

Center for Primary Care and Public Health University of Lausanne Lausanne Switzerland

Center for Translational Immunology University Medical Center Utrecht and Princess Máxima Center for Pediatric Oncology Utrecht The Netherlands

Department of Chemistry Graduate School of Sciences and Engineering Koç University Istanbul Turkey

Department of Electron Microscopy Molecular Biology The Cyprus School of Molecular Medicine The Cyprus Institute of Neurology and Genetics Nicosia Cyprus

Department of Glycobiotechnology Institute of Chemistry Slovak Academy of Sciences Bratislava Slovakia

Department of Medical Chemistry and Biochemistry Faculty of Medicine and Dentistry Palacky University Olomouc Czech Republic

Department of Medical Oncology Midland Regional Hospital Tullamore St James's Hospital Dublin Ireland

Department of Molecular Cell Biology Institute for Cancer Research Oslo University Hospital Oslo Norway

Department of Neurology Erasmus MC Rotterdam The Netherlands

Department of Physical Chemistry Vinča Institute of Nuclear Sciences University of Belgrade Belgrade Serbia

Ephraim Katzir Department of Biotechnology Engineering ORT Braude College Karmiel Israel

Functional Proteomics Laboratory Centro Nacional De Biotecnología Madrid Spain

Human Genetics Department Instituto Nacional De Saúde Dr Ricardo Jorge Lisboa Portugal and Centre for Toxicogenomics and Human Health Universidade Nova De Lisboa Lisbon Portugal

Immunologix Laboratories Tampa FL USA

Immunology Division LCMN Germans Trias 1 Pujol University Hospital and Research Institute Campus Can Ruti Badalona and Department of Cellular Biology Physiology and Immunology Universitat Autònoma De Barcelona Cerdanyola Del Vallès Spain

Institute of Cancer Therapeutics University of Bradford Bradford UK

Institute of Medical Chemistry Faculty of Medicine University of Belgrade Belgrade Serbia

Institute of Natural Sciences Department of Biochemistry Ege University Izmir Turkey

Laboratory for Analytical Chemistry Department of Chemistry and Biochemistry Faculty of Food Technology and Biotechnology University of Zagreb Zagreb Croatia

Laboratory for Biology of Reproduction Institute for the Application of Nuclear Energy INEP University of Belgrade Belgrade Serbia

Materials Research and Technology Department Luxembourg Institute of Science and Technology Belvaux Luxembourg

Medical Faculty Department of Biomedical Sciences Chair of Medical Biology and Department of Structural Biology Medical University of Lodz Łódź Poland

Medisiina D 6 A B University of Turku Turku Finland

Mutagenesis Unit Institute for Medical Research and Occupational Health Zagreb Croatia

Neuroimmunology and Neuroinflammation Group Instituto De Investigación Biomédica De Málaga IBIMA UGC Neurociencias Hospital Regional Universitario De Málaga Malaga Spain

Proteomics Department Institute of Cellular Biology and Pathology N Simionescu of the Romanian Academy Bucharest Romania

Research Institute for Chromatography Kortrijk Belgium

Translational Metabolic Laboratory Department of Laboratory Medicine Radboud Institute of Molecular Life Sciences Radboud University Medical Center Nijmegen The Netherlands

Université Grenoble Alpes CEA Inserm IRIG BGE Grenoble France

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