The need for new biomarkers to support personalized healthcare, has fostered numerous proteomic innovations. Still, a number of challenges remain. One is the preponderance of high abundance proteins and, concurrently in targeted proteomic workflows, efficiency and consistency in quantifying target biomarker peptides from different sample cohorts. This is in part due to the changing landscape of proteins/peptides not associated with the selected biomarker targets. A solution for both these challenges is now available through a suite of products called NRicher™. This bead-based technology is derived from experience of over 10 years at the forefront of manufacturing beads (i.e., ionic, hydrophobic, hydrogen bonding, aromatic, polymeric) with differential proteome binding properties. NRicher™ now offers a solution that stands out in its simplicity and versatility. The NRicher™ workflow follows a bead-based bind/wash protocol using a standard microfuge to separate the buffer solutions from the beads. The NRicher™-derived sub-proteomes (different for each product application) are bound to the beads, followed by Bead-Assisted Sample Prep (BASP™), performing reduction, alkylation and digestion directly on the bead-bound proteome. The use of detergents or denaturants is not required. Once complete, the derived peptides seamlessly integrate to the LC-MS analysis. NRicher™ products were evaluated by LC-MS/MS for specific purposes, including: enrichment of low abundance human serum proteins for improving overall coverage, and for targeted proteomics – decreasing LC gradient time to improve productivity. Comparisons to pooled neat (without enrichment) serum are evaluated.
Using NRicher™, enrichment of low abundance protein targets (<1 µg/ml) reach MS signal levels typically observed for mid-abundance proteins (1-100 µg/ml) and with less background noise, effectively suited for quantitation of target peptide signals. Consequently, NRicher™ sub-proteome enrichment can minimize acquisition time, collectively improving overall throughput, cost, and productivity. For these reasons, sample prep workflows combining NRicher™ sub-proteome enrichment with on-bead digestion will become especially desirable.
We now introduce the NRicher™ Knowledgebase with a purpose so that researchers can enrich for particular protein biomarker(s) of interest. The Knowledgebase contains over 2000 proteins with corresponding relative signal intensities that can help select for the best NRicher™ bead/method combination for the biomarker(s) of interest; all freely accessible on a non-confidential basis. The Knowledgebase identifies and annotates > 100 prospective biofluid biomarkers associated with disease conditions, with references. Also, annotation of >200 soluble membrane proteins, derived from ectodomain shedding, a common dysregulated disease mechanism, and an important source of precision biomarkers are curated. Finally, we describe strategies to use the Knowledgebase to compartmentalize and profile systemic chronic inflammation, and associated diseases.