Highly Multiplexed Proteomic Platform for Biomarker Discovery, Diagnostics, and Therapeutics

Abstract

Progression from health to disease is accompanied by complex changes in protein expression in both the circulation and affected tissues. Large-scale comparative interrogation of the human proteome can offer insights into disease biology as well as lead to the discovery of new biomarkers for diagnostics, new targets for therapeutics, and can identify patients most likely to benefit from treatment. Although genomic studies provide an increasingly sharper understanding of basic biological and pathobiological processes, they ultimately only offer a prediction of relative disease risk, whereas proteins offer an immediate assessment of "real-time" health and disease status. We have recently developed a new proteomic technology, based on modified aptamers, for biomarker discovery that is capable of simultaneously measuring more than a thousand proteins from small volumes of biological samples such as plasma, tissues, or cells. Our technology is enabled by SOMAmers (Slow Off-rate Modified Aptamers), a new class of protein binding reagents that contain chemically modified nucleotides that greatly expand the physicochemical diversity of nucleic acid-based ligands. Such modifications introduce functional groups that are absent in natural nucleic acids but are often found in protein-protein, small molecule-protein, and antibody-antigen interactions. The use of these modifications expands the range of possible targets for SELEX (Systematic Evolution of Ligands by EXponential Enrichment), results in improved binding properties, and facilitates selection of SOMAmers with slow dissociation rates. Our assay works by transforming protein concentrations in a mixture into a corresponding DNA signature, which is then quantified on current commercial DNA microarray platforms. In essence, we take advantage of the dual nature of SOMAmers as both folded binding entities with defined shapes and unique nucleic acid sequences recognizable by specific hybridization probes. Currently, our assay is capable of simultaneously measuring 1,030 proteins, extending to sub-pM detection limits, an average dynamic range of each analyte in the assay of > 3 logs, an overall dynamic range of at least 7 logs, and a throughput of one million analytes per week. Our collection includes SOMAmers that specifically recognize most of the complement cascade proteins. We have used this assay to identify potential biomarkers in a range of diseases such as malignancies, cardiovascular disorders, and inflammatory conditions. In this chapter, we describe the application of our technology to discovering large-scale protein expression changes associated with chronic kidney disease and non-small cell lung cancer. With this new proteomics technology-which is fast, economical, highly scalable, and flexible--we now have a powerful tool that enables whole-proteome proteomics, biomarker discovery, and advancing the next generation of evidence-based, "personalized" diagnostics and therapeutics.

Validation of a blood protein signature for non-small cell lung cancer

“…We also reported that this analysis was confounded by large scale differences between the study sites. This observation motivated a series of studies investigating the effect of sample handling on biomarker discovery [22,23]. The result of these studies was the development of a set of SMVs which allowed us to quantify the magnitude of confounding pre-analytical variation introduced by sample collection differences.For example, the cytoplasmic molecular chaperone HSP90 is a well established lung cancer marker and therapeutic target.…”

Non-helical DNA Triplex Forms a Unique Aptamer Scaffold for High Affinity Recognition of Nerve Growth Factor

“…Such chemically modified aptamers are particularly well-suited to selection schemes that favor long-lived complexes; hence, we refer to this class of aptamers as SOMAmers (slow off-rate modified aptamers). SOMAmers have transformed the landscape of proteomics, leading to a sensitive and highly multiplexed platform with applications in biomarker discovery and early-stage disease detection (De Groote et al, 2013;Gold et al, 2010Gold et al, , 2012Loffredo et al, 2013;Lollo et al, 2014;Mehan et al, 2012Mehan et al, , 2013Ostroff et al, 2010Ostroff et al, , 2012, as well as sandwich-based single analyte detection assays (Ochsner et al, , 2014, rapid histochemistry tools (Gupta et al, 2011), and therapeutic applications (Gelinas et al, 2014;Gupta et al, 2014).…”

Identification of ssDNA aptamers specific to clinical isolates of <italic>Streptococcus mutans</italic> strains with different cariogenicity

“…SELEX has been successfully applied to a variety of targets including proteins [44], small peptides [45], metal ions [46], and small organic molecules [47]. In basic medical research, this method has been used for drug screening [48][49][50][51], clinical diagnosis [52], and treatment [53]. Whether the library is enriched or not is one of the important indicators of SELEX selection success or failure.…”