Enriching low-abundant proteins from complex biological matrices is crucial in numerous proteomic workflows. This article outlines an immunoprecipitation method compatible with Quantum-Si’s next-generation protein sequencing workflow.
This method involves an on-bead digestion protocol that eliminates the sample elution step, proceeding directly to the preparation of digested peptide libraries. The viability of this method is demonstrated by selectively enriching IL-6 via immunoprecipitation from human serum, followed by protein sequencing and identification on Platinum®.
Introduction
The human blood proteome is a complex mixture of low and high-abundant proteins with a concentration spectrum spanning over 10 orders of magnitude.1 Low-abundant proteins, like growth factors, chemokines, and cytokines, are of significant interest in biomedical research as they are indicators of various disease states.
For example, Interleukin-6 (IL-6) is a cytokine that has various important roles within immune system activation. In healthy individuals, IL-6 is found in the blood at moderately low concentrations (1—5 pg/mL). However, the production of IL-6 undergoes a rapid escalation in response to tissue injuries, infections, and other immune challenges, thereby establishing it as an early disease biomarker.2
Investigating low-abundant proteins in biological samples necessitates specialized methods for enriching and concentrating the proteins of interest to ensure effective analysis. Immunoprecipitation (IP) is a widely employed method in biochemistry and molecular biology. The fundamental principle of IP is effective and straightforward.
Antibodies, designed to specifically target the protein of interest, are initially immobilized on a solid matrix (like agarose or magnetic beads). Monoclonal antibodies that exhibit high specificity and affinity for the target protein are usually used to guarantee effective antigen-antibody interactions.
The sample containing proteins of interest (such as a cell lysate or a biological fluid like serum) is subsequently incubated with the antibody-coated beads.
The antibodies selectively bind to the target protein, creating an antibody-protein complex. In successive washing steps, unwanted contaminants and proteins are eliminated, leaving the specific target protein(s) entrapped with the beads. The captured protein is then eluted from the antibody-protein complex, readying it for later downstream protein evaluation.
After isolating the immunoprecipitated protein, the identity of the proteins in the sample can be evaluated utilizing proteomic analysis methods, including immunoassays and mass spectrometry. However, these methods necessitate sophisticated equipment and analysis or cannot offer information from amino-acid level sequencing.
Next-generation protein sequencing with Quantum-Si’s Platinum® is an alternative method for identifying proteins following immunoprecipitation with single amino acid resolution in a simple benchtop workflow. This innovative technique involves digesting the isolated proteins into peptide fragments and attaching them to macromolecular linkers at the C-terminus.
These peptides are immobilized on Quantum-Si’s semiconductor chip and probed with dye-labeled N-terminal amino acid (NAA) recognizers. This process yields unique pulsing patterns distinguished by characteristic kinetic properties.
Aminopeptidases in solution successively remove individual NAAs, unveiling the NAA of subsequent amino acids for identification.
The collected fluorescence intensity, lifetime, and kinetic data are then analyzed using cloud-based software to determine the peptide sequence and corresponding protein.
To highlight the effectiveness of this method, Quantum-Si enriched IL-6 from human serum with immunoprecipitation and used Quantum-Si’s Platinum instrument for next-generation protein sequencing of the IL-6 peptide libraries.
Sequencing of the digested peptide libraries yielded single-molecule peptide traces that corresponded with peptides from IL-6, successfully enriching and identifying the protein from human serum.
These findings underscore the complementary nature of immunoprecipitation of target proteins from complex samples and subsequent protein identification analysis on Quantum-Si’s Platinum instrument.
References and further reading
- Anderson NL, Anderson NG. The human plasma proteome: history, character, and diagnostic prospects [published correction appears in Mol Cell Proteomics. 2003 Jan;2(1):50]. Mol Cell Proteomics. 2002;1(11):845-867. doi:10.1074/mcp.r200007-mcp200
- Jones SA, Takeuchi T, Aletaha D, Smolen J, Choy EH, McInnes I. Interleukin 6: The biology behind the therapy. Considerations in Medicine. 2018 Nov 1;2(1):2-6
About Quantum-SI
Inspired by Ion Torrent’s success at shrinking next-generation sequencing technology into a benchtop instrument, Jonathan Rothberg founded Quantum-Si™ to bring the same semiconductor technology to protein sequencing with the launch of the Platinum® Next-Generation Protein Sequencer™.
That was in Guilford, CT, back in 2013. Fast forward to today and we now have over 1,000 patents issued and applications pending, plus a groundbreaking single-molecule protein sequencing technology platform, the Platinum.
Along the way, we solved critical challenges around sensitive and unambiguous amino acid detection, blending biology, chemistry, and semiconductor technology to help biologists see what other approaches cannot deliver. We also set the stage for a revolution in how scientists understand biology and build new treatments for disease by making single molecule protein sequencing accessible to every lab everywhere.
We are now entering a new phase of our development as a company. Starting with an initial public offering in June 2021 (QSI on the NASDAQ) and continuing with a new product development and operations facility in San Diego, CA, in 2022, we have entered a period of rapid growth. Through this expansion, we will be able to fuel a new era of biology, the post-genomic era, where biologists accelerate basic scientific insight and biomedical advances through the power of next-generation protein sequencing.
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