Why Abundance-Only Readouts Miss Immune Function

How protein organisation reveals hidden mechanisms

A playbook showing that protein organization reveals hidden mechanisms

Scenario: when expression does not explain function

You are profiling lymphoma B cell lines to evaluate therapeutic targets.

Protein abundance analysis shows that key therapeutic targets such as CD40 are present at high levels on the lymphoma cells. In fact, CD40 expression is comparable between some of the lymphoma B cells and healthy B cells.

CD40 has been explored as a therapeutic target in lymphoma, but responses have not been consistent. Paradoxical, it has been shown that healthy B cells and Burkitt’s lymphoma cells respond differently to CD40 stimulation, healthy B cells proliferate, while some lymphoma cells go into apoptosis.

Figure legend: The costimulatory protein CD40 is an interesting target in lymphoma thanks to its high expression on cancer B cells.

A challenge for interpretation:

If the target is present at similar levels, why do the biological and therapeutic responses differ between the cells?

The abundance measurement can lead to:

incorrect therapeutic predictions
misinterpreted disease mechanisms

When to look beyond abundance

Consider expanding the analysis beyond abundance when:

✓ Expression profiles are similar but function differs
✓ Target abundance does not explain therapeutic response
✓ Known markers do not fully explain the biology

The Missing Dimension: Protein Organisation

Protein function is not only regulated by what proteins are expressed, but also by how these proteins are arranged. This includes the formation of functional protein complexes, clusters of proteins and exclusion of regulatory proteins.

Hence, to resolve hidden functional heterogeneity, analyse:

Protein clustering
Colocalisation
Protein domains

What abundance alone does not show in Burkitt’s lymphoma B cells

CD40 is highly expressed by both healthy B cells and 3 Burkitt’s lymphoma cell lines: Raji, Daudi and Ramos. CD40 levels are comparable across healthy B cells and Daudi and Ramos lymphoma B cells.

However, analysis of CD40 organization reveals a different layer of regulation. Cell surface architecture analysis showed that:

✓ Each cell type has a distinct cell surface architecture
✓ Membrane protein domains partially overlap, but differ in composition
✓ CD40 organisation differs between healthy B cells and lymphoma cells

In lymphoma cells, CD40 becomes less associated with CD20-tetraspanin-rich membrane domains.

This difference is not visible from abundance measurements alone.

Figure legend: CD40 colocalizes with CD20-tetraspanin-rich domains in healthy B cells but not in Burkitt’s lymphoma cell lines.

Why this matters

Tetraspanins are known regulators of membrane organisation and receptor signalling.

Changes in membrane organisation may influence:

✓ Receptor interactions
✓ Signalling behaviour
✓ Functional response to antibody targeting

This provides a possible explanation for why cells with similar CD40 abundance may still respond differently.

What the data supports

Directly shown:

✓ Similar CD40 abundance across healthy and lymphoma B cells
✓ Distinct membrane organisation patterns between cells
✓ Altered CD40 organisation in lymphoma cells

Suggested:

✓ Protein organisation may contribute to altered functional behaviour and therapeutic response

Key concept

Protein abundance defines which proteins are present.

Protein organisation reveals how those proteins are arranged to function.

Takeaway

Cells can appear similar by protein expression while functioning differently. Protein organisation can reveal biological differences that abundance alone cannot detect.

Explore further about the technology and its application here:
Protein Interactomics by Proximity Networks - Pixelgen Technologies

Application note “Molecular Pixelation reveals cancer-specific protein patterns of individual cells” link:
https://www.pixelgen.com/application-notes/

GO BEYOND PROTEIN EXPRESSION

Measure more than which proteins are present. Map how cell surface proteins are organized, connected, and spatially arranged across single cells.

Pixelgen’s Proximity Network Assay brings nanoscale protein interactomics to high-throughput immune cell research, helping teams uncover biology that abundance-based methods can miss.

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