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Abstract BACKGROUND:
Complex biofluids such as human plasma encompass a concentration range of over ten orders of magnitude, with albumin alone comprising approximately 50% of total protein mass, thereby masking lower-abundance biomarkers during LC-MS/MS analysis. Traditional depletion approaches often require extensive handling, large volumes, or specialized instrumentation, limiting throughput and reproducibility. The Nanotrap® Protein Enrichment Affinity Kit (PEAK) addresses these challenges by employing three proprietary magnetic hydrogel particle chemistries (Nanotrap® Protein Particles A, Nanotrap® Protein B, and Nanotrap® Protein C) to selectively capture, concentrate, and release target proteins and peptides while concurrently reducing the presence of high abundance proteins all within a single 45-minute workflow. This universal enrichment platform is compatible with multiple commercial digestion chemistries—including Promega Rapid-Digest Trypsin/Lys-C, Pierce In-Solution Tryptic Digestion and Guanidination Kit, PreOmics® iST 8X Kit, and Thermo Scientific™ SMART Digest™ Trypsin Kit—without requiring instrument-specific hardware.
METHODS:
K2EDTA human plasma samples were thawed and centrifuged briefly using low-speed centrifugation. 50 µL plasma samples were diluted four-fold in Nanotrap® Buffer 4. Samples were then incubated with Nanotrap Protein A, B, and C Particles according to the Combined-, 2-, or 3-Particle methods for 30 minutes at room temperature with gentle agitation. After magnetic separation and two washes with HPLC-grade water, bound proteins were chemically reduced (TCEP) and alkylated with iodoacetamide before proteolysis. Four digestion workflows were evaluated in parallel: Promega Rapid-Digest Trypsin/Lys-C (2 hours at 70°C), Pierce™ In-Solution Tryptic Digestion and Guanidination, PreOmics iST 8X Kit with on-column cleanup, and Thermo Scientific SMART Digest Trypsin Kit with magnetic bulk resin; each required ≤45 min of additional processing. Peptides were desalted using ZipTip™. LC-MS/MS analysis was performed on a Thermo Scientific™ Orbitrap Exploris™ 480 in data-dependent acquisition mode.
RESULTS:
Integration of Nanotrap® PEAK with each digestion chemistry yielded substantial improvements in proteome depth compared to unenriched controls. Using the Promega Rapid-Digest workflow, enrichment delivered 2.5- to 4.0-fold increases in unique protein identifications, with the 3-Particle Method consistently achieving the highest coverage. Experiments with the Pierce In-Solution kit demonstrated between 2.2- to 4.0-fold improvements across the Combined, 2-, and 3-Particle methods. The Thermo SMART Digest workflow yielded 1.1- to 2.4-fold enhancements relative to neat plasma, identifying a total of 1,058 unique proteins with a single-particle enrichment strategy. PreOmics iST workflows corroborated these trends, producing 2.0- to 4.3-fold gains across gradient lengths and digestion formats. Crucially, the Combined Particle Method depleted albumin to <3 % of total peptide signal—corresponding to >97 % removal—enabling enhanced detection of low-abundance analytes. Gel-based evaluation via SYPRO Ruby staining confirmed robust exclusion of the 65 kDa albumin band by each particle chemistry while preserving a diverse range of protein profiles. Technical reproducibility of identified protein groups was assessed across six biological replicates. Unenriched plasma exhibited a median CV of 13% across replicates, whereas manual Nanotrap PEAK protocols produced a median CV of 8.4% and semi-automated enrichment on the KingFisher™ Apex System achieved a median CV of 2.3%. Rank-abundance and dynamic range analyses further demonstrated that both manual and automated workflows extended protein detection into lower-abundance ranges and improved proteome coverage.
CONCLUSION:
Nanotrap PEAK enables highly reproducible protein enrichment from human plasma while effectively depleting high-abundance proteins such as albumin, resulting in significantly improved proteome coverage. Across multiple workflows and digestion chemistries, Nanotrap PEAK demonstrated consistent albumin depletion (>97%) and enhanced detection of low-abundance proteins, enabling up to a 4.3-fold increase in unique protein identifications. The platform showed excellent technical reproducibility, with median coefficients of variation as low as 2.3% using semi-automated protocols. Nanotrap PEAK’s compatibility with diverse LC-MS/MS platforms and digestion kits, combined with rapid processing and scalable formats – makes it a powerful tool for biomarker discovery and quantitative plasma proteomics.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
