Home > Technical Resources > Case Studies > NRicher™ Bead Platform Provides Unique Sub-Proteome Biases And Fit For Purpose Opportunities for Targeted LC-MS Quantification

NRicher™ Bead Platform Provides Unique Sub-Proteome Biases And Fit For Purpose Opportunities for Targeted LC-MS Quantification

Background

Proteomic innovation continues to drive the need for new biomarkers to support personalized healthcare. For biofluid proteomics, a constant challenge is to observe and quantify low to mid-abundance sub-proteomes as the preponderance of high abundance proteins (>top 100) can obscure analysis. For untargeted proteomics, coverage has become an important goal, with many different depletion and enrichment products and strategies available, the choice often depending upon the goals and nature of the investigation. Thus, many products and methods, including many supplied by BSG, are dedicated to enhancing proteome coverage, especially from biofluids like blood and its derivatives, red cells and serum/plasma. Nevertheless, for targeted proteomics, efficiency and consistency in quantifying target peptides from different sample cohorts presents signal to noise challenges due to the changing landscape of proteins/peptides not associated with the selected targets.

So for targeted proteomics, there are different sample prep demands. Target proteins of interest may come from different sources including those identified through discovery efforts, gene expression, or curated from public domain databases and publications. Consequently, targeted proteomics is emerging as a compelling alternative to traditional antibody-based methods for protein biomarkers. Once identified, the next necessary step is the subsequent quantification of the pre-identified targets in robust cost-effective LC-MS workflows, so as to establish validity and potential clinical utility as biomarkers.

Nevertheless, the precise detection of specific target peptides remains a significant challenge. Co-eluting peptides from non-targets can generate ions similar to the targeted peptide, as well as suppress ionization. These interferences lead to challenges in distinguishing and accurately quantifying the intended target signal amid background non-target noise. This is especially problematic for low abundance peptides from complex biological samples like blood plasma, the predominant sample type in clinical use. A simple sample prep that can enrich specific protein(s) of interest in targeted proteomic workflows is therefore highly desirable.

Solution – The NRicher™ Platform and Knowledgebase

NRicher™ is a bead-based technology 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. Several commercial products based on singular bead surface features have been derived from the fundamental NRicher™ bead chemistry platform, most notably AlbuVoid™ and HemoVoid™. NRicher™ consists of consumable chemically derived porous beads, and an adaptability to bead cocktails. NRicher™ products do not require any specialized instruments, can be processed using a standard microfuge, with protocols suitable for automated liquid handlers. All necessary features for high-throughput cost-effective targeted proteomics.

Function of the Knowledgebase

Over 2000 proteins observed with corresponding relative signal intensities

Find your protein(s) of interest, and corresponding bead/methods to best enrich for protein(s) of interest, free to review, accessible on a non-confidential basis

Annotation of >200 soluble membrane proteins, derived from ectodomain shedding, a common dysregulated disease mechanism, and important source of precision biomarkers

Annotation of >100 prospective biofluid biomarkers associated with disease conditions


Annotation of >100 prospective biofluid biomarkers associated with disease conditions, enriched using NRicher™ - representative examples





Each NRicher™ bead chemistry provides differential sub-proteome coverage. Note that AlbuVoid & HemoVoid are derived from NRicher™ bead chemistry platform.

Plasma proteins that are unique to each enriched preparation (absent from neat)

Notable results:
<5% common to all 4 bead preps

15% common between AlbuVoid™ PLUS and HemoVoid™

11% common between AlbuVoid™ and NRicher™ MX

Each NRicher™ bead chemistry enriches low abundance biomarker proteins at remarkably different LC-MS signals (semi-quantitative), ideal for targeted and multiplex proteomics – some representative examples



Comparison of NRicher™ Apo vs. Neat (untreated) pooled human serum Multiplex Targeted Peptides Demonstrate Substantially Improved Spectral Signals After Enrichment

A Selection of Peptide Levels with Variations That Represent Common Functional Level Reporting Features



ApoE is associated noncovalently with lipids as part of lipoprotein complexes. Single residue gene level variant substitutions alter interactions with specific lipoprotein sub-populations. ApoE4 is widely recognized as the major genetic risk factor for AD, while ApoE2 may be protective.



Both apoC-I and C-II appear in the circulation predominantly as full length native proteins along with minor truncated proteoforms. Ratios of these proteoforms are under investigation in atherosclerosis and coronary heart disease (CHD).

Why Choose NRicher™
● Not derived from immuno-affinity: NRicher™ products are not species-specific.
● Cost-efficient: There's no need for an investment in high-end specialized equipment; a standard laboratory microfuge will suffice.
● Through proper selection of NRicher™ beads, multiplex peptide targets from specific functional regions, such as spanning regions of proteolysis, truncations, amino acid variants or other PTMs can be explored in depth. This extends research beyond strict abundance level quantitation, to more discretionary functional levels. Protein level enrichment can serve to enhance signal to noise of the functionalities associated with post-translational modifications (PTMs).
● Streamlined Analysis: Through the optional use of bead cocktails, NRicher™ products serve virtually all applications, starting with sample volumes as low as 25 µl. To further enhance enrichments, enrichment protocols can be refined through optimal load, and buffer conditions.
● Use the Knowledgebase of >2000 proteins to find the optimal NRicher™ products for your biomarker(s) of interest.

All NRicher™ beads are available in standard list price 10 or 50 prep kits, as well as bulk quantities. Please inquire at sales@biotechsupportgroup.com.

Download Knowledgebase




For more information, download posters first presented at HUPO World Congress 2024




Learn more about the NRicher™ family specific product line