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Complex biofluids like human plasma contain a vast range of protein concentrations. This variability presents both opportunities and challenges. With a rich composition of proteins and peptides, plasma samples are a valuable source for proteomic studies. However, highly abundant proteins like albumin often mask the identification of low-abundance proteins, which may have high biological and clinical relevance. Reducing the dynamic range of plasma proteins could open new avenues in biomarker discovery and the study of disease mechanisms.
Bead-based enrichment and depletion of proteins are some solutions to enhance the detection of low-abundance proteins. They involve the isolation and concentration of specific proteins from complex samples for detailed analysis. Bead-based enrichment uses small, solid particles (beads) coated with specific binders that can selectively bind to proteins or peptides of interest.
Here's an overview of how bead-based enrichment works and its applications:
The Principles of Bead-Based Enrichment
1. Binding: The sample containing a mixture of proteins is incubated with the coated beads, allowing the accessed proteins to bind to the beads via the specific interaction between the target and the surface.
2. Washing: The beads are then washed to remove non-specifically bound material, leaving only the specifically bound proteins.
3. Lysis: Accessed proteins are denatured, reduced, and alkylated by combining them with a LYSE reagent and incubating them in a thermal shaker for ten minutes.
4. Digestion: The proteins are then digested into peptides using a proteolytic enzyme, typically trypsin, under optimized conditions for complete digestion.
5. Purification: Digested peptides are cleaned to remove any remaining contaminants that could interfere with mass spectrometry analysis. This can be done using solid-phase extraction (SPE) or other purification techniques.
6. Mass spectrometry analysis: Cleaned-up peptides are dried and reconstituted in an appropriate solvent prior to injection into the mass spectrometer for analysis.
Bead-based enrichment offers a highly specific and efficient way to isolate proteins from complex samples. It enhances the sensitivity and specificity of downstream analytical techniques such as mass spectrometry, making it a valuable tool in proteomics research and clinical diagnostics.
Interested in Enrichment Kits?
Bead-based enrichment can involve using various kits and workflows, including our ENRICH-iST. The ENRICH-iST kit by PreOmics offers a solution to the challenges of accessing low-abundant proteins in plasma and serum samples. Traditional workflows can be time-consuming, involving extensive hands-on time, and consequently, more prone to errors due to variability in sample handling and interference of contaminants, for example. Likewise, the impracticality of automation, lack of flexibility, and inability to produce unbiased results impede the application for large sample cohorts.
The ENRICH-iST kit provides a fast, easy-to-use, standardized, and automatable protocol for preparing plasma and serum samples by enriching low-abundance proteins onto paramagnetic beads, maintaining proteome coverage. This kit ensures scalability, improved efficiency, exceptional reproducibility, low coefficient of variations (CVs), and a quick processing time of only 5 hours from raw sample to pure peptides. It is compatible with human samples and other mammalian species like mice, rats, pigs, or dogs. The ease of use, compatibility with lab automation systems, and reduced processing time make it ideal for high-throughput handling large sample cohorts efficiently.
If you would like to learn more, read our blog on targeting low-abundance proteins in plasma and serum proteomics. Otherwise, contact a member of the PreOmics team today with any queries.
