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22 | JULY 2016 | GENengnews.com | Genetic Engineering & Biotechnology News Dominic Zichi, Sheri Wilcox, Jefrey Walker, and Nebojsa Janjic Proteomics is the comprehensive study of protein expression. The goal of proteomics today is to enable a large number of protein measurements on a large number of samples, analogous to now routine large-scale genom- ic measurements. The SOMAscan ® assay is helping realize this goal. Mass spectrometry (MS) and antibody- based techniques are dominant proteomic technologies, but both suffer from challeng- es for large-scale protein profling. Because most proteomic studies are aimed at com- plex mixtures (blood, for example) the rela- tively high abundance of a few proteins can lead to biased results. We refer to this as the "albumin/IgG problem," since albumin and globulins account for over 93% of proteins in blood. Discovery MS can identify large numbers of proteins and provide relative changes in abundance for small numbers of samples, but limited sensitivity results in biases to- ward high-abundance proteins. MS also suf- fers from low throughput, relatively poor reproducibility, and limited dynamic range. Antibody-based technologies are ubiq- uitous in the feld. These techniques are able to detect low abundance proteins (sub- nanomolar). Antibodies are also adaptable to measurements in complex matrices, but individual antibodies can be subject to nonspecifc binding. Even high affnity an- tibodies will bind nontarget proteins in a matrix where the target is dramatically less abundant. ELISAs overcome this issue by using two antibodies in a sandwich format; an initial antibody captures the target and a second antibody detects. The specifcity of each an- tibody helps isolate the target among abun- dant nontarget proteins. While the sandwich method proved to be a helpful advance, it no longer meets the needs of proteomic re- searchers wishing to perform highly multi- plexed, large-scale profiling studies. The main reason is that ELISAs do not scale well in a highly multiplexed assay due to nonspe- cifc binding of both capture and detection antibodies. A Better Way to Profle Proteins The SOMAscan assay allows for simul- taneous detection of thousands of proteins (multiplexing) while assaying large numbers of samples (throughput). In its current ver- sion, the assay can measure 1,310 protein targets from as little as 65 µL sample (serum, plasma, cerebrospinal fuid, tissue, cells, and other complex matrices). The low sample volume requirement maximizes the utility of precious clinical samples. In addition to this high-level of multiplexing, the assay covers a large dynamic range detecting proteins from femtomolar to micromolar concentrations. A Novel Reagent The SOMAscan assay uses a unique set of protein-binding reagents aimed at con- formational epitopes to achieve large-scale multiplexing and throughput. SOMAmer ® reagents (Slow Off-rate Modifed Aptamers) are single-stranded DNA constructs contain- ing modifed functional groups mimicking amino acid side chains involved in protein binding. Like high-affnity monoclonal an- tibodies, these reagents allow for affnity capture with outstanding specifcity to the given protein epitope. Since recognition is directed to conformational epitopes, binding to shared epitopes across highly conserved proteins can occur. SOMAmer reagents derive their high af- fnity and specifcity from functional group and shape complementarity to protein epit- opes. The novel protein-like side chains in- troduced into these reagents allow the SO- MAmer DNA backbone to adopt unique secondary and tertiary structures and facili- tate tight binding to protein targets. 1,2 The complementary interactions with protein targets and the unique SOMAmer structures adopted by these molecules are illustrated (Figure 1). Each SOMAmer reagent is selected through an iterative in vitro evolution method known as SELEX (Systematic Evo- lution of Ligands by EXponential enrich- ment). Selection pressure enriches for both high affnity and slow off-rates that are key to SOMAscan assay performance. Once selected, the sequence is chemically synthe- sized using standard solid-phase techniques, generating a uniform and reproducible cus- tomized reagent. SOMAscan Assay for Large-Scale Proteomics The SOMAscan assay is a two-catch as- say that overcomes the "albumin/IgG prob- lem" by combining the initial binding with "kinetic specifcity enrichment" to measure low abundance analytes in complex matri- ces. This format allows for two elements of specifcity, analogous to an antibody sand- wich assay, using a single reagent. Because it relies on a single affnity reagent and disso- ciation kinetics (slow for specifc and fast for non-specifc interactions), the SOMAscan assay scales easily to thousands of simulta- Elucidating Biology with a Highly Multiplexed Proteomics Platform Measuring Proteins on a Massive Scale OMICS Assay Tutorial Figure 1. Co-crystal structures of three SOMAmer reagents, highlighting the exquisite interactions between the reagents and their targets, the unique structures adopted by these reagents, the modifcations (in red), and the extensive surface areas of each interaction. The protein in the crystal structure is represented by a surface and the SOMAmer reagent as a wire-frame with a backbone trace. The secondary structure for the SOMAmer reagent is displayed below the crystal structure. Figure 3. Volcano plot comparing ~4,000 protein measurements for DMD patients compared to control subjects. Each symbol indicates a distinct protein, with blue and red symbols indicating proteins with higher or lower concentrations in DMD patients compared to controls, above a statistical threshold. Figure 2. An overview of the SOMAscan assay workfow.