Western blot is one of the most widely applied techniques for protein identification. However, attaining reproducible and reliable results depends on the availability of quality antibodies with validated specificity for the target protein. In Western blot analysis, differentiating between differences in amino acid sequences and isoforms can be challenging if the protein size is similar.
To address these challenges, there is a growing demand for non-antibody-based methods. This article outlines an in-gel protein digestion and peptide extraction technique compatible with Quantum-Si’s library preparation and protein sequencing workflows.
This technique facilitates the isolation and enrichment of single proteins from complex mixtures utilizing SDS-PAGE, enabling their subsequent identification on Quantum-Si’s Platinum® next-generation protein sequencing instrument.
Introduction
Separating and enriching complex samples is a crucial aspect of proteomic workflows. Gel electrophoresis is a relatively simple technique for sample enrichment. Of the various gel electrophoresis methodologies, sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is widely employed for protein mixtures.
During SDS-PAGE, an electric field is applied to denatured proteins bound to the anionic detergent SDS. This causes the proteins to migrate through a crosslinked polyacrylamide gel matrix. This migration segregates the proteins by their mass-to-charge ratio and length, yielding distinct protein bands that are resolved in the gel upon staining.
Although SDS-PAGE facilitates the separation and enrichment of complex samples, further downstream procedures are needed for the identification of the isolated components.
Western blot is a widely employed technique used to identify specific proteins after they are separated by SDS-PAGE. In this method, proteins are transported from the gel to a nitrocellulose membrane, followed by blocking and subsequent incubation with a primary antibody designed to target the protein of interest.
A secondary enzyme-conjugated antibody is subsequently employed to bind to the primary antibody, generating a signal that can be pictured on a western blot imager for protein identification. While this approach is the most commonly utilized antibody-based method, the availability of correctly validated antibodies devoid of off-target binding remains challenging. This issue results in wasted time, money, reagent waste, and inaccurate data.1
Exploring alternative antibody-free techniques for sensitive protein identification can significantly improve research productivity and minimize waste.
One promising method of identifying pre-separated components (without the utilization of antibodies) is next-generation protein sequencing with Quantum-Si’s Platinum workflow.2 This innovative method involves excising proteins from the SDS-PAGE gel, digesting them into peptide fragments, and attaching them to macromolecular linkers at the C-terminus.
The peptides are subsequently immobilized on Quantum-Si’s semiconductor chip and probed with dye-labeled N-terminal amino acid (NAA) recognizers, producing distinguishing pulsing patterns with characteristic kinetic properties. Aminopeptidases in solution sequentially remove individual NAAs, revealing subsequent amino acids for identification.
A real-time collection of fluorescence lifetime, intensity, and kinetic data is conducted. These are then analyzed using Cloud-based software to determine the peptide sequence and corresponding protein.
Quantum-Si has established an in-gel digestion technique compatible with Quantum-Si's downstream library preparation and sequencing workflows to leverage the advantages of separating complex mixtures through gel electrophoresis and the protein identification abilities of Platinum.
The in-gel digestion method outlined here is similar to procedures utilized in other proteomic workflows.3 To highlight the effectiveness of this method, Quantum-Si generated in-gel digested peptide libraries utilizing the protein CDNF (Cerebral Dopamine Neurotrophic Factor) as a model protein.
Sequencing of the in-gel digested peptide libraries yielded individual molecule peptide traces that corresponded with peptides from CDNF, facilitating the accurate identification of the protein.
These findings underscore the compatibility of the in-gel digestion procedure with Quantum-Si’s library preparation and sequencing workflow, indicating its potential to separate and enrich more complex sample mixtures for the resolution of variations in amino acid sequence.
References and further reading
- Baker M. Reproducibility crisis: Blame it on the antibodies. Nature. 2015;521(7552):274 276. doi:10.1038/521274a
- Reed BD, Meyer MJ, Abramzon V, et al. Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device. Science. 2022;378(6616):186-192. doi:10.1126/science.abo7651
- Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc. 2006;1(6):2856-2860. doi:10.1038/nprot.2006.468
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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