The Impact of Carbon Microbead Crosslinker Composition on Proteomics Workflows

Proteomics labs increasingly push LC–MS systems to their limits, yet sample prep is often constrained by media that are suboptimal for today’s harsh conditions, complex matrices, or high-sensitivity PTM analysis

All‑carbon microbeads with tunable pore size and surface chemistry offer a way to decouple capacity from “stickiness” so you can match the bead to the biology, not the other way around. For detailed comparison tables and protocol guidance on Vinyl (referred to as NanoPak-C-VL) vs Acrylate (referred to as NanoPak-C-AE) crosslinked All Carbon Microbeads in proteomics, see our Technical Application Note in the Library & Resources section.

Why media choice still limits proteomics

Most proteomics workflows run into a similar set of practical challenges.

• Cleaning up samples without losing useful analytes. Salts, detergents, chaotropes, and other additives must be removed before LC–MS, but each cleanup step risks losing fragile or low‑abundance analytes along with contaminants.

• Detecting rare peptides and PTMs against a vast background. Modified or low‑abundance peptides are often present at trace levels compared with their unmodified counterparts. Thus, these peptides need to be selectively enriched without a significant excess of background peptides.

• Handling intact and membrane proteins under demanding conditions. Large proteins and membrane complexes may not enter standard pore structures (10-20 nanometers). Further, the high pH or strong organic solvents can damage conventional silica‑based media.

In each scenario, two parameters matter the most:

• Pore size, which sets the largest size of molecules that can reach the inside surface.

• Surface chemistry affects how water-repellent the material is, how much unwanted sticking happens, and how stable it is in different acids, bases, and liquids.

All carbon microbeads with vinyl-based (NanoPak-C-VL) and acrylate (NanoPak-C-AE) crosslinkers were developed to help proteomics scientists confidently select the right chemistry for their specific workflows, emphasizing the importance of matching bead chemistry to workflow needs.

Two chemistries, two proteomics roles

Both NanoPak-C-VL and NanoPak-C-AE microbeads can be made with large pores (>100 nm), so intact

proteins and peptides can access the internal surface in either case. What differentiates them is the backbone chemistry and the resulting interaction profile with the analytes (see Table).

NanoPak‑C‑VL microbeads are suitable for workflows that require exposure to harsh conditions (high alkaline pH) or require the removal of sticky proteins or detergents. Some potential applications include top-down proteomics, processing membrane assemblies, and the cleanup of hydrophobic serum components.

Key use‑cases include:

• Top-down intact protein fractionation. Traditional chromatographic pores often exclude large proteins (those above 50 kDa), resulting in low capacity and poor resolution. NanoPak-C-VL beads, with their 100–200 nm pores, allow antibodies and membrane proteins to access the particle interior and interact with the aromatic surface. This design delivers strong retention and efficient hydrophobicity-based fractionation, significantly improving sample preparation for mass spectrometry (MS) workflows.

  
  

• High‑pH reversed‑phase fractionation (“Spider”‑style workflows). Vinyl-crosslinked (NanoPak-C-VL) microbeads are durable across a wide range of alkaline pH levels allowing multiple high-pH fractionation processes and effective cleaning with strong bases. For example, one can fractionate tryptic peptides using reversed-phase (RP) methods at high alkaline pH (pH 10–12), they can complement traditional low-pH LC-MS separation methods, thereby enhancing proteome coverage.

  
  

• Depletion of high-abundance, sticky components and detergents. Serum albumin, IgG, and membrane proteins can dominate total protein mass and obscure low-abundance biomarkers. NanoPak-C-VL microbead’s very hydrophobic, aromatic surface firmly retains these “stickier” proteins, allowing more soluble, lower‑abundance targets to pass through for downstream analysis.

The same principle applies to non-volatile detergents such as Triton X-100 and SDS, which contain aromatic or highly hydrophobic moieties that bind to NanoPak-C-VL microbeads and can be removed before use in LC–MS systems.​

• Media Regeneration. Because of its wide pH window and solvent tolerance, NanoPak-C-VL microbeads can be regenerated with a strong base or high organic solvent, which is particularly valuable in high-throughput or core facility settings where columns and cartridges must withstand repeated harsh cleaning cycles.​

NanoPak-C-AE microbeads are appropriate for workflows that require gentle handling of peptides. NanoPak‑C‑AE microbeads’ surface is neither too hydrophobic (“greasy”) nor polar. The polar ester groups present on acrylate crosslinkers, along with the hydrophobic graphite, confer orthogonal selectivity relative to standard C18, a purely hydrophobic phase. They offer gentle handling of peptides while still enabling effective cleanup and fractionation. Potential applications include bottom-up proteomics, phosphoproteomics, and glycoproteomics.

Typical applications include:

• Trap peptide solid phase extraction. NanoPak-C-AE microbeads could be used in high-load solid-phase extraction columns to concentrate peptides. It limits nonspecific peptide adsorption and exhibits sufficient interaction and retention of the peptides of interest. For bottom-up proteomics, the challenge is often not retention, but over‑retention of non-specific peptides and other components present in the sample that masks true enrichment of the peptides of interest. These concentrated peptides can then be introduced into LC–MS systems.

  
  

• Post-Translation modification (PTM) enrichment with a lower hydrophobic background. When enriching for phosphopeptides or glycopeptides, non-phosphorylated hydrophobic peptides can dominate binding in highly hydrophobic media, reducing selectivity and dynamic range. NanoPak-C-AE microbeads are still hydrophobic enough to engage peptides. Still, they are less prone to indiscriminate binding, which lowers the hydrophobic background and improves the adequate enrichment of modified species.​

• Guard peptide solid phase extraction. This type of extraction can deplete “greasy” peptides. In membrane protein digest analysis, highly hydrophobic transmembrane peptides can overload or foul LC columns downstream. NanoPak-C-AE microbeads selectively retain these ultra-hydrophobic peptides under low-organic conditions. The more soluble peptides pass through—essentially a negative-selection step that purifies the digest before the sample is introduced into high-resolution LS-MS systems.​

An easy method to check both these microbeads in your lab.

The following straightforward evaluation experiment can be conducted by proteomics teams interested in using these chemistries. No modification of the core LC–MS setup is required. You can use this test to evaluate which bead chemistry is best suited to your proteomics needs before committing to more advanced column setups or automation.

• Start with solid-phase extraction and desalting of a model digest. Pack a small quantity of either NanoPak-C-VL or NanoPak-C-AE All Carbon Microbeads into pipette tips or spin columns. Use these tips of columns to process a standard sample (e.g., tryptic digest of bovine serum albumin (BSA)). Employ a simple aqueous acid load-and-wash step, followed by elution with 50–80% acetonitrile, and compare the recovery, background, and chromatographic separation of the eluates.

In summary, your decision to choose between NanoPak-C-VL and NanoPak-C-AE microbeads comes down to picking the right surface chemistry for the pH and solvent conditions required by your specific proteomics approach—whether that’s top-down or bottom-up proteomics, post-translational modification (PTM), or membrane proteins. These microbeads with adjustable pore sizes provide a flexible solution for desalting, fractionation, depletion, and detergent removal using a single material type that can be adapted to fit your workflow.

For detailed comparison tables, pH and cleaning limits, and step-by-step workflows (including high‑pH fractionation, Spider‑style setups, scout columns, and negative‑mode depletion), refer to the complete user guide in the Library & Resources section of the Millennial Scientific website.

Get in touch with us to discuss how we can support your chromatography separation needs. For more information or to request samples, please email us at inquiry@millennialscientific.com, call us at 855 388 2800, or fill in our online form.

References

User Guide: All‑Carbon Microbeads for Proteomics – Differentiating Crosslinker Chemistry for Top‑Down and Bottom‑Up Workflows, 2026.