Sponsored content brought to you by

Humans have approximately 20,000 human protein-coding genes, and the proteins they express span a concentration range of ~12 logs. Yet only 0.1% of proteins contribute to 99% of the total plasma protein mass.¹ The remaining 1% contains highly relevant, low-abundance biomarkers, including cytokines, chemokines, interferons, and proteins released by early-stage tumors. Detecting these crucial biomarkers requires a very sensitive protein assay. A new end-to-end, NGS-based approach from Illumina facilitates consistent, reproducible detection.

Profiling proteins broadens understanding of cellular function and molecular processes. Indeed, >58% of labs conducting transcriptomics research and >54% of labs conducting genomics research also use proteomics to bridge the genotype and phenotype gap.²

Integrating proteomics

Integrating proteomics into an omic mix massively increases dynamic complexity. Unlike the genome, the transcriptome and proteome are in a constant state of flux. Furthermore, proteins are folded into a vast number of possible conformations and undergo post-translational chemical modifications to produce the final functional unit. Add in splice variants and  somatic recombination and the intricacy becomes apparent.³

Many low abundance, highly relevant circulating proteins are below the nanomolar range and require enrichment or extremely high specificity for accurate detection without false positives. A new NGS-based method, Illumina Protein Prep, uses aptamers to provide the necessitated high sensitivity and specificity to broaden the biological discovery reach.

Aptamers broaden the context

Mass spectrometry (MS) and antibody-based methods are considered legacy standards for high-throughput proteomics despite their significant limitations. For example, antibody-based platforms suffer from batch-to-batch variability, high cost of antibody production, and relatively low chemical stability.^4,5

Furthermore, proteins cannot be directly amplified for measurement, rendering legacy methods less scalable than those for studying nucleic acids. Newer affinity-based approaches address this challenge by using oligonucleotide aptamers that allow sequencing, enabling NGS readout and all the resultant benefits, including scalability, streamlined workflows, and multiomics endeavors.

The term “aptamer” is derived from the combination of the Latin word aptus (“to fit”) and the Greek word meros (“part”). First described in 1990, aptamers are short single-stranded oligonucleotides that bind with high affinity and specificity to proteins and peptides.^6,7

Aptamers recognize a protein based on 3D structure and are capable of distinguishing between closely related molecules, targets containing different functional groups, or even an amino acid mutation. Ten-fold smaller than antibodies, aptamers facilitate multiplexing capabilities and increase access to targets, without cross-reactivity. Production of these synthetic molecules reliably delivers consistent, reproducible results resulting in low coefficient of variation (CV) values for aptamer-based protein assays compared to antibody-based methods.^8,9,10 Even minute changes in protein expression can be profiled accurately allowing access to that highly-relevant 1% protein mass.

Aptamer design has progressed. A versatile chemistry technology endowed nucleotides with protein-like functional groups that mimic amino acid side chains. This greatly expanded the repertoire of accessible targets in highly multiplexed proteomics arrays. New strategies to select aptamers with very slow off-rates are used for these Slow Off-rate Modified Aptamers (SOMAmer^®).¹⁰

SOMAmers are orthogonally validated and can be effectively used to simultaneously detect thousands of proteins in multiplex discovery platforms. A vast range of additional functional groups can be added at any desired position in the oligonucleotide sequence, making SOMAmers the most versatile reagent in proteomics.¹¹

An end-to-end NGS-based approach

Illumina Protein Prep’s end-to-end, NGS-based approach empowers discovery and relative quantification of 9.5K unique human proteins, covering over 200 biological pathways in a single plasma or serum sample. The high-throughput proteomics assay uses

SOMAmer reagents for protein capture, providing femtomolar sensitivity with a 10-log dynamic range, unlimited multiplexing capabilities, and excellent reproducibility with <6% CV.¹²

The SOMAmers provide a high degree of shape matching to the protein target, allowing discernment between nearly identical proteins. The comprehensive panel targets human proteins across a diverse set of pathophysiological processes, including cancer, inflammation, immunology, and cardiometabolic function, and covers secreted, intracellular, and extracellular molecular targets such as receptors, kinases, growth factors, and hormones. Even low abundance markers like interferon can be detected.

The Illumina Protein Prep solution uses a streamlined, integrated workflow spanning protein capture, library preparation, sequencing, analysis, and data visualization to provide results in less than 2.5 days with ~4 hours of hands-on time. Sequencing data analysis tools contain the fully integrated DRAGEN™ Protein Quantification pipeline for secondary data analysis that includes NGS and proteomic assay-specific normalization methods and the tertiary analysis and visualizations in Illumina Connected Multiomics.

Proteomics makes a difference

For earlier detection of eight common cancer types, a large collaborative study used CancerSEEK, a blood test that assessed levels of circulating proteins and mutations in cell-free DNA. In a 1005-patient cohort targeting eight nonmetastatic, clinically-detected cancers CancerSEEK tests were positive in a median of 70% of all cancer types. Sensitivities ranged from 69-98% with specificity greater than 99%.¹²

Building on these encouraging results, a subsequent study enrolled over 10,000 participants to determine if the presence of cancer could be identified at a stage when treatment was more likely to be successful. DETECT-A (Detecting cancers Earlier Through Elective mutation-based blood Collection and Testing) built on CancerSEEK but used pre-defined thresholds along with a confirmation test to enhance specificity. The authors confidently concluded that, at present, a minimally invasive blood test could be safely used to detect several cancer types in patients not previously diagnosed.¹⁵

“Leveraging the initial 6K Illumina assay, we investigated the reproducibility and predictive power of circulating proteins in early cancer detection,” said Kamel Lahouel, PhD, Assistant Professor at TGen, who was involved in the second study. “Our analysis demonstrated excellent reproducibility, with both intraplate and interplate correlations reaching R² ~0.97. Importantly, using a four-fold cross-validation approach—training on three plates and testing on the fourth—we consistently achieved high sensitivity at high specificity, with an average AUC of 0.96.”

In each round of the 4-fold cross-validation, the research team identified the top 40 most discriminating proteins, which indicates that the selected proteins generalized well across plates. Notably, 10 proteins were consistently selected across all folds, underscoring their potential as robust predictive biomarkers.

Lahouel emphasized that “This highlights the robustness of the selected features and is promising for their reproducibility and generalizability in future runs. With most tested cancers in early stages and 11 cancer types represented in the cohort (N=334 samples), such an assay can be a powerful tool when integrated into multi-cancer early detection tests, and also for protein discovery and features selection.”

Expand the discovery scope 

Genomics, epigenomics, and transcriptomics provide indirect measurements of cellular states but may not accurately reflect the corresponding complex and dynamic protein changes that can occur with time or signaling events. Proteomics can complete the puzzle with important functional insights as well as validation of omics data.

Parallelizing proteomics, genomics, and transcriptomics studies opens the doors to scalability and a new level of biological knowledge. Compatibility with NovaSeq™ 6000 and NovaSeq X Systems affords multiomic access across the entire central dogma of molecular biology on a single platform.

While genomics and transcriptomics show what could happen, proteomics reveals what is actually happening at a molecular level. Proteomics can connect genotype to phenotype, facilitating the elucidation of disease mechanisms, identification of novel drug targets, discovery of biomarkers, and potentially accelerate personalized medicine research.

The proteome’s dynamic and complex nature requires exceptionally sensitive, scalable, and high-throughput methodologies, like Illumina Protein Prep, to realize its full potential in discovery.

References

1. Tirumalai RS et al. Characterization of the low molecular weight human serum proteome. Mol Cell Proteomics. 2003; 2(10): 1096-1103. doi: 10.1074/mcp.M300031-MCP200.
2. Percepta Associates, Inc. Global Multiomics Practices Market Research. 2022.
3. Aebersold R et al. How many human proteoforms are there? Nat Chem Biol. 2018 Feb 14; 14(3):206-214. doi: 10.1038/nchembio.2576.
4. Baker M. Reproducibility crisis: Blame it on the antibodies. Nature. 2015; 521(7552): 274-276. doi: 10.1038/521274a.
5. Bradbury A, Plückthun A. Reproducibility: Standardize antibodies used in research. Nature. 2015; 518(7537): 27-29. doi: 10.1038/518027a.
6. Ellington A, Szostak J. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990). doi: org/10.1038/346818a0.
7. Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug 3 ;249(4968): 505-10. doi: 10.1126/science.2200121.
8. Eldjarn GH et al. Large-scale plasma proteomics comparisons through genetics and disease associations [published correction appears in Nature. 2024 Jun; 630(8015): E3. doi: 10.1038/s41586-024-07549-z]. Nature. 2023; 622(7982): 348-358. doi: 10.1038/s41586-023-06563-x.
9. Rooney MR et al. Plasma proteomic comparisons change as coverage expands for SomaLogic and Olink. Preprint. medRxiv. 2024;2024.07.11.24310161. Published 2024 Jul 12. doi: 10.1101/2024.07.11.24310161.
10. Gold L et al. (2010) Aptamer-Based Multiplexed Proteomic Technology for Biomarker Discovery. PLoS ONE 5(12): e15004. doi: 10.1371/journal.pone.0015004.
11. Thiviyanathan V, Gorenstein DG. Aptamers and the next generation of diagnostic reagents. Proteomics Clin Appl. 2012 Dec; 6(11-12):563-73. doi: 10.1002/prca.201200042.
12. Cohen JD et al. Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science. 2018 Feb 23; 359(6378): 926-930. doi: 10.1126/science.aar3247.
13. Illumina. Illumina Protein Prep data sheet. Published October 22, 2024. Accessed January 21, 2025.
14. Olink. Olink Explore HT. Accessed February 7, 2025.
15. Lennon AM et al. Feasibility of blood testing combined with PET-CT to screen for cancer and guide intervention. Science. 2020 Jul 3; 369(6499): eabb9601. doi: 10.1126/science.abb9601.

To learn more, download our NEW ebook illumina.com.

For Research Use Only. Not for use in diagnostic procedures. M-AMR-01784