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NRicher™ C

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Product Code: NRICHER-C

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NRicher™ C

Enrichment of Complement Cascade Related Proteins

Consumable chemically derived beads, species agnostic as they are not derived from antibodies

Enrich Complement Cascade related proteins from sera/plasma or cell lysates from both animals and humans, >90% Albumin removal

Does not require any specialized instruments, just a standard microfuge

Bead format suitable for automation compatibility, please inquire

On-Bead digestion for LC-MS analysis, or optional elution for any functional, enzymatic, or immunoassay analysis

The complement cascade is a major component of the immune system that provides powerful host surveillance and protection from invading microbes. It also exerts an important influence on the adaptive immune response by acting synergistically with antibodies as well as promoting B- and T-cell stimulation. The intracellularly active complement system—the complosome, has been shown to play a critical function in regulating T cell responses, cell physiology (such as metabolism), and inflammatory disease processes. Complement dysregulation is implicated in chronic diseases ranging from age-related macular degeneration to neurological & cardiovascular disease, as well as cancer. In acute inflammatory conditions, hyperactive complement may predispose individuals to adverse outcomes, as suspected in hospitalized Covid-19 patients.

The complement system consists of over 50 circulating and membrane proteins, comprising about 5% of the total protein mass in plasma. Most complement proteins circulate in blood as inactive precursors (zymogens), that when triggered, become activated through proteolytic cascades. Although textbooks describe three activation pathways (classical, lectin & alternative), largely under-appreciated is Complement’s evolutionarily conserved link to coagulation to eliminate damaged tissues. Complement can activate platelets and contribute to hemostasis in response to injury. Conversely, complement can be activated from proteolytic enzymes derived from coagulation and fibrinolysis that can cleave both C3 and C5 leading to amplification, and final function. As a result of complement activation, many outcomes are produced including: opsonization of pathogens or damaged-self cells to enhance phagocytosis; production of anaphylatoxins C3a & C5a; recruitment of leukocytes to the inflammatory site; and the terminating end of the cascade – assembly of the membrane attack complex (MAC) on the cell surface.

Click Here To View NRicher™ C Product Sheet

The terminating end of the complement cascade is derived from the C3 Convertase proteolytic product - C5b, which engages the sequential recruitment of C6, C7, C8, and C9, assembling the membrane attack complex (MAC). Also known as the “terminal complement complex”, it results from the coordination of C5b-7 insertion in the membrane, which then captures C8, inducing polymerization of a C9 ring – to as many as 18, C9’s per pore. Terminal MACs perforate the cell membrane of the invading pathogen or target cell, and when a sufficient number of MAC pores form, the cell dies by osmotic lysis. Sub-lytic doses of MAC however, induce dramatically different effects than lytic doses, including adherence, aggregation, chemotaxis, cell division, and extracellular vesicle release.

In such a critical juncture, the complement system must maintain a delicate balance between activation and inhibition to allow activation when necessary to counteract infectious agents or modified self/host tissues, while concurrently protecting healthy self/host tissue. This protection is achieved systemically through the concerted action of regulators and inhibitors ensuring cell and tissue integrity essential for normal and healthy well-being.  NRicher™ C provides enrichment of all Complement-related proteins so as to interrogate functional features derived from proteolysis (split products) or gene variants. Specific tryptic peptides that report functional regions are especially noteworthy after NRicher™ C enrichment; for example - C3a, C4BPB, CFB (Ba fragment), peptides have approximately >10X total spectral signal compared to neat (not enriched) serum. Multiplex targeted proteomics can therefore investigate a more complete picture of Complement activation and regulation using NRicher™ C, than would be possible with immunoassay measurements.

Product	Size	Total serum/plasma samples processed	Item No.
NRicher™ C	10 Preps	10 x (25-50) µl samples	NRCO-10
NRicher™ C	50 Preps	50 x (25-50) µl samples	NRCO-50

References

New Jersey Commission On Science, Innovation And Technology Phase II Catalyst Research Grant Awarded to Biotech Support Group

Biotech Support Group announces receipt of a NEW JERSEY COMMISSION ON SCIENCE, INNOVATION AND TECHNOLOGY Phase II Catalyst Research Award of $40,000. The Research Award entitled “ Evaluating the NRicher™ Bead Platform for Targeted LC-MS Quantification” will be conducted at The Rutgers Biological Mass Spectrometry Facility. This award follows from the Catalyst Phase I Award demonstrating simple workflows that generate low abundance enrichment of sub-proteomes suitable for quantitative plasma proteomics.

Swapna LS, Stevens GC, Sardinha-Silva A, Hu LZ, Brand V, Fusca DD, et al. (2024 ) ToxoNet: A high confidence map of protein-protein interactions in Toxoplasma gondii. PLoS Comput Biol 20(6): e1012208. https://doi.org/10.1371/ journal.pcbi.1012208

The article states we used affinity beads (NuGel PROspector) to pre-enrich Toxoplasma gondii lysate to capture five distinct subproteomes. [Note: NuGel PROspector beads are now part of the NRicher™ platform.]. When comparing the 5 different subproteomes, there is clearly different selection biases amongst the 5 surface chemistries. Also, many of the proteins observed from the NRicher™ beads, were not observed in the Ion exchange fractions demonstrating the importance of combining different modes (ionic, hydrophobic, etc.) of separation to alter selection properties, and consequently improving overall proteome coverage.

Efficiencies gained in targeted serum proteomics using NRicher Beads – simplified and diversified workflows for sub-proteome and biomarker enrichment – Poster HUPO World Congress 2024

After NRicher™ sample prep, target peptides have highly enhanced spectral signal. NRicher™ sub-proteome enrichment can minimize acquisition time, collectively improving overall throughput, cost, and productivity. Specific target peptides that report functional PTMs and amino acid variant regions promise insights and potential multiplex biomarkers for disease.

https://www.biotechsupportgroup.com/v/vspfiles/templates/257/pdf/HUPO%202024%20Efficiencies%20Gained.pdf

Wan, C., Borgeson, B., Phanse, S. et al. Panorama of ancient metazoan macromolecular complexes. Nature 525, 339–344 (2015). hXps://doi.org/10.1038/nature14877

Six different NRicher™ beads (described with an old tradename PROspector) were used as an enrichment step in the overall workflow; about twice the number of observations and annotations became possible. This further validates that the sub-proteome bias characteristics of the NRicher™ surface chemistry platform can simplify complex proteomes into enriched sub-proteomes with efficiencies suitable for deep functional proteome characterization.

Whitepaper - NRicher™: A Low Abundance Proteome Enrichment Platform With Seamless Integration of On-Bead Digestion

The NRicher™ Advantage is described: • Consumable chemically derived NuGel™ beads, species agnostic as they are not derived from antibodies • Does not require any specialized instruments, just a standard microfuge • Use of bead cocktails allows for one, rather than multiple LC-MS analyses • Functionally active sub-proteomes after separations, for any orthogonal functional, enzymatic, or immunoassay analysis

https://www.biotechsupportgroup.com/v/vspfiles/templates/257/pdf/BiotechSupportGroup-NRicher-Whitepaper.pdf

NRicher : Family Specific Enrichment For Targeted Proteomics – Poster US HUPO 2024

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 peptides from different sample cohorts. This is in part due to the changing landscape of proteins/peptides not associated with the selected targets. A solution for both these challenges is now available through a suite of products called NRicher .

https://www.biotechsupportgroup.com/v/vspfiles/templates/257/pdf/NRicher%20poster%20small.pdf