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The Deliverome Project

Platform overview

Targeted delivery requires surface proteins specific to the right cells, present at the right abundance on those cells, and that get cargo to the appropriate place within the cell. We will measure these properties across tissues and cell types through a combination of proteomics and functional genomics technologies.

Protein abundance

Most targeting decisions rely on existing RNA atlases — but what matters for delivery is protein on the cell surface, and the two routinely disagree. We're developing two complementary approaches for measuring surface protein abundance directly: (1) global DIA mass spectrometry, which gives absolute copy numbers across the proteome, and (2) glycoprotein enrichment, which trades absolute counts for deeper coverage of the glycoproteins commonly found on the cell surface.

Diagram of the abundance platform. On the left, surface proteins are measured across many human tissues and cell types — liver, kidney, heart, lung, spleen, pancreas, brain, primary cells and more. On the right are two workflows. Global proteomics digests whole samples with trypsin and reads them by LC-MS/MS to give absolute protein abundance. A glycoprotein-enrichment platform first captures glycosylated proteins on beads coated with glycan-reactive functional groups, then digests and reads them by LC-MS/MS to give relative abundance, providing differential measurements between tissues for low-abundance membrane proteins.Diagram of the abundance platform. On the left, surface proteins are measured across many human tissues and cell types — liver, kidney, heart, lung, spleen, pancreas, brain, primary cells and more. On the right are two workflows. Global proteomics digests whole samples with trypsin and reads them by LC-MS/MS to give absolute protein abundance. A glycoprotein-enrichment platform first captures glycosylated proteins on beads coated with glycan-reactive functional groups, then digests and reads them by LC-MS/MS to give relative abundance, providing differential measurements between tissues for low-abundance membrane proteins.

Internalization

A surface protein is only useful for delivery if it brings cargo inside. We'll rank internalization across the surfaceome with two complementary approaches: enrichment-based mass spectrometry measures which endogenous proteins get internalized, and pooled functional-genomics screens can capture surface proteins proteomics methods may miss. Together they give a ranked, surfaceome-wide view of which proteins ferry cargo into cells.

Proteomics

Surface-biotinylation proteomics assay for internalization. Cell-surface proteins are labelled with biotin, then cells are warmed to 37°C so some proteins internalize. A cell-impermeable reducing agent strips biotin from proteins remaining on the surface, leaving only internalized proteins tagged. A streptavidin pull-down followed by mass-spectrometry quantitation ranks proteins by how much each internalized.

Functional genomics

Pooled functional-genomics screen for internalization. Each cell carries a DNA barcode and displays a surface construct built from SpyCatcher and SpyTag, which normalizes for expression level, plus an ALFATag bound by a cleavable ALFATag nanobody. After warming to 37°C, a reducing agent or protease removes tags left on the surface so remaining signal reflects internalized construct. Cells are sorted by normalized internalization and their barcodes sequenced to rank surface proteins across the pool.

Trafficking

Internalized cargo doesn't always end up where you need it — endosome, lysosome, or back out to the surface. The route determines whether a payload works. The same pooled screens let us map where each surface protein sends its cargo next.

Trafficking assay using split reporters. Barcoded cells display a SpyTag/ALFATag surface construct bound by an anti-epitope nanobody, together with an anti-endosomal marker and an anti-lysosomal marker that each carry one half of a split reporter. After warming to 37°C, cargo that traffics to the endosome reconstitutes the endosomal reporter, shown as a purple compartment, while cargo that reaches the lysosome reconstitutes the lysosomal reporter, shown as a dark-red compartment — revealing each surface protein's destination.

Delivery

The atlas is only useful if it predicts what gets delivered. Functional delivery assays close the loop. They connect abundance, internalization, and trafficking to the question that matters: does a prototypical payload reach its target and do its job?

Functional delivery assay in three stages. Format: binders of varying affinity and format are tested. Modality: each binder is paired with a payload — for example siRNA or a toxin — on a tagged targeting construct. Functional outcome: an siRNA payload is read out as knockdown of a target gene, and a toxin payload is read out as reduced cell fitness — connecting abundance, internalization and trafficking to whether a payload actually reaches its target and works.