Mass Spec Roundup: Vesicles, Tumors, and Doping…

Traffic Control: Mapping Protein Transport Through the Golgi

Researchers at the Hong Kong University of Science and Technology (HKUST) have combined vesicle reconstitution with quantitative mass spectrometry to map how adaptor protein complexes AP-1 and AP-4 mediate the transport of proteins through the cell’s secretory pathway. The work, published in PNAS, identifies previously unknown cargo clients and regulatory factors involved in vesicular trafficking.

Using an in vitro vesicle formation assay and label-free quantitative proteomics, Prof. Yusong Guo’s team pinpointed CAB45 as an AP-1–dependent cargo and ATRAP (angiotensin II type I receptor–associated protein) as an AP-4–dependent cargo. The researchers also uncovered two cytosolic accessory proteins – WDR44 and PRRC1 – that regulate AP-4–mediated transport from the trans-Golgi network (TGN).

Electron microscopy and proteomic analyses revealed that AP-4 recognizes a tyrosine-based motif on ATRAP to enable its export from the Golgi. When WDR44 or PRRC1 were disrupted, AP-4 cargos such as ATG9A and ATRAP accumulated abnormally at the TGN, impairing downstream cellular processes including autophagy.

“These results not only deepen our understanding of AP-1 and AP-4 functions in secretory trafficking but also provide a powerful methodological toolkit for systematically dissecting the mechanisms of specific accessory factors,” said Guo in a press release.

Nanobody Tool Disrupts RNA Condensates in Kidney Cancer

A study in Nature Communications has revealed that in translocation renal cell carcinoma (tRCC), RNA assembles into liquid-like condensates that activate tumor-driving genes. Breaking down these RNA-built hubs, scientists found, can halt the disease at its molecular source.

The team at Texas A&M University Health Science Center combined CRISPR-based tagging, SLAM-seq to track nascent transcripts, CUT&Tag and RIP-seq to map fusion protein–RNA interactions, and quantitative mass spectrometry–based proteomics to catalogue proteins recruited into the condensates. These analyses identified PSPC1, an RNA-binding protein, as a stabilizing factor that enhances tumor growth.

To dismantle the structures, the researchers engineered a nanobody-based chemogenetic switch that selectively binds to the fusion proteins and, upon activation, dissolves the RNA condensates. The intervention stopped cancer growth in cultured tRCC cells and in mice.

“RNA itself is not just a passive messenger, but an active player that helps build these condensates,” said senior author Yun Huang. Co-author Yubin Zhou added that targeting condensate formation “opens the door to therapies that are much more precise and potentially less toxic.”

Because similar fusion proteins occur in other pediatric tumors, the work provides a blueprint for exploiting RNA’s structural roles – and for cutting power to cancer’s hidden molecular control hubs.

Spot Check

A team at the German Sport University Cologne has developed a simplified liquid chromatography–mass spectrometry (LC–MS³) method for detecting anabolic steroid esters in dried blood spots (DBS), offering a faster, more practical alternative for anti-doping analysis. The study, published in the Journal of Mass Spectrometry, demonstrates that 17 native steroid esters – including testosterone, nandrolone, and boldenone derivatives – can be sensitively and selectively detected without chemical derivatisation.

The researchers used a Vanquish UHPLC system coupled to a Thermo Stellar mass spectrometer operated in MS³ mode with collision-induced (CID) and higher-energy collisional dissociation (HCD) activation. The hybrid quadrupole–multipole–ion trap setup enabled sub-nanogram per millilitre detection limits and recoveries of 43–85 percent across both cellulose- and polymer-based sampling devices.

In validation trials, all esters were reproducibly detected at or below 0.1 ng/mL, and authentic samples from a volunteer administered 80 mg of oral testosterone undecanoate confirmed real-world applicability. Compared with conventional isotope-ratio MS approaches used for urinary steroid analysis, the method reduces analytical complexity and turnaround time.

“The approach demonstrates that DBS, combined with MS³ detection, can provide a robust and sensitive tool for doping control without the need for derivatisation,” the authors conclude. The technique may also be extended to other prohibited compound classes, enhancing the efficiency of modern anti-doping laboratories.

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