Affinity Purification Mass Spectrometry on the Orbitrap–Astral Mass Spectrometer Enables High-Throughput Protein–Protein Interaction Mapping

Classical proteomics experiments offer high-throughput
protein
quantification but lack direct evidence of the spatial organization
of the proteome, including protein–protein interaction (PPIs)
networks. While affinity purification mass spectrometry (AP-MS) is
the method of choice for generating these networks, technological
impediments have stymied the throughput of AP-MS sample collection
and therefore constrained the rate and scale of experiments that can
be performed. Here, we build on advances in mass spectrometry hardware
that have rendered high-flow liquid chromatography separations a viable
solution for faster throughput quantitative proteomics. We describe
our methodology using the Orbitrap–Astral mass spectrometer
with 7 min, high-flow separations to analyze 216 AP-MS samples in
∼29 h. We show that the ion-focusing advancements, rapid mass
analysis, and sensitive ion detection facilitate narrow-bin data-independent
acquisition on a chromatographically practical timescale. Further,
we highlight several aspects of state-of-the-art confidence-scoring
software that warrant reinvestigation given the analytical characteristics
of the Orbitrap–Astral mass spectrometer through comparisons
with an enrichment-based thresholding technique. With our data, we
generated an interaction map between 998 human proteins and 59 viral
proteins. These results hold promise in expediting the throughput
of AP-MS experiments, enabling more high-powered PPI studies.