To traverse this barrier, the qPCR readout detection was recently upgraded to an innovative play on next-generation sequencing (NGS) to give rise to the PEA-NGS technology, as described by Darmanisetal.(82). proximity extension assay, aptamer, single-molecule array, technology comparison, technology advances Abbreviations:CV, coefficient of variation; ECL, electrochemiluminescence; ELISA, enzyme-linked immunosorbent assay; LED, light-emitting diode; LC, liquid chromatography; MS, Mass spectrometry; MSD, meso scale discovery; NGS, next-generation sequencing; NPX, normalized protein expression; PCR, polymerase chain reaction; PE, phycoerythrin; PEA, proximity extension assay; Simoa, single-molecule array; SOMAmer, slow off-rate modified aptamer; ssDNA, single-stranded DNA; TPA, tripropylamine == Graphical Abstract == == Highlights == Immunoassay-based multiplex proteomics methods measure 1000+ proteins in biofluids. Multiplex protein measurements alleviate demands on time, cost, and sample volume. This guide helps researchers choose the most suitable tool for their applications. == In Brief == Probing the human plasma proteome is attractive for biomarker and drug target discovery. Recent breakthroughs in multiplex proteomics technologies enable the simultaneous and sensitive quantification of thousands of proteins in biofluids. We provide a comprehensive guide to the methodologies, performance, advantages, and disadvantages of established and emerging technologies for the multiplexed ultrasensitive measurement of proteins. Gaining knowledge on these innovations is crucial for choosing the right multiplexed proteomics tool to critically complement traditional proteomics methods. Studying the JTK4 proteome is central to understanding the functional units of a cell. Proteins are involved in a breadth of biological processes, from apoptosis, to cellular checkpoints, to inflammation to name a few, all of which are pertinent to diseases ranging from cancer, to neurodegenerative, cardiovascular, and infectious diseases (1,2,3). Studying the alterations in protein expression, secretion, and interactions in biological fluids can elucidate disease pathways and discover novel therapeutic targets and noninvasive biomarkers (4,5). Recent breakthroughs in immunoassay-based multiplex protein technologies now allow for the simultaneous quantification of hundreds to thousands of proteins in a single assay. However, applying these novel platforms to map the circulating disease proteome requires superb technical sensitivity to detect scarce disease-associated proteins, excellent reproducibility, and cost-effective means for robustly powered validation studies and clinical trials in the arduous effort of bringing a novel drug or biomarker to the clinic. When it comes to clinical practice, the technical demands further increase with added expectations of realistic sample volume requirement and turnaround time. Equipped with a growing array of ultrasensitive multiplex proteomics technologies, selecting the right tool for the question at hand can be daunting without first Calcipotriol acquiring considerable knowledge surrounding the variety of technologies. We herein provide a comprehensive guide of the technological principles, performance, strengths, and weaknesses of commercial immunoassay-based technologies for Calcipotriol the multiplexed ultrasensitive measurement of proteins in human biofluids. The principles, advantages, and drawbacks of some of the technologies described in this Review have been reviewed elsewhere (6,7,8,9). In these cases, we provide updates on the rapidly evolving field, where advancements in the multiplexing capability and analytical prowess of the technologies are unveiled on a yearly basis, to enable researchers to discover more of the human proteome. == Challenges in Protein Detection and Measurement Calcipotriol == Unlike the rapid advances in whole genome sequencing, traditional proteomics methods have lagged behind in terms of improving throughput and sensitivity for measuring proteins in biofluids at the proteome-wide scale. In comparison with the genome, which consists of about 20,000 genes, the proteome is far more complex with an estimated close to one million proteins, after accounting for alternative splicing and posttranslational modifications (1). It is therefore unsurprising that it takes decades to develop the incredibly powerful technical methods required to Calcipotriol chart the complete human proteome, let alone unravel how protein expression dynamically changes in various diseases (10). Mass spectrometry (MS) Calcipotriol remains the core tool for proteomics efforts, with the two main methodologies for the wide detection of proteins being bottom-up and top-down MS (reviewed elsewhere) (11,12,13). Bottom-up MS is 100-fold more sensitive than the latter method and is favored for discovery-based proteomics for its broader coverage and higher throughput (11). Though capable of detecting up to several thousand proteins in a single liquid chromatography (LC)-MS/MS experiment, the method may hold bias toward high-abundance proteins and delivers less robust detection of low-abundance proteins (13). When it comes to using complex biological matrices such as human serum or plasma, the performance of MS techniques further deteriorates with even targeted methods typically unable to detect proteins below low g/ml or high ng/ml levels without extensive sample fractionation or enrichment to decrease the sample complexity (14). To this end, the utility of MS for identifying novel cancer biomarkers or drug targets is hindered by insufficient resolution of the human proteome (15). The typical dynamic range of LC MS/MS.
