Episodic Transport of Protein Aggregates Achieves a Positive Size Selectivity in Aggresome Formation

To generate this dataset, Dynabeads Protein G beads (Thermo Fisher, #10004D) were used to immunoprecipitate different forms of purified AgDD proteins through an HA-tag on the AgDD after incubating AgDD proteins in the Xenopus egg extract. AgDD samples used in the study include AgDD aggregates formed in the extract (AgDDXE), in the physiological buffer (AgDDpb) and the unaggregated control (AgDDsol). Two batches of actin-depolymerized CSF extracts were freshly prepared in parallel and used for experiments as repeats. Proteins immunoprecipitated by the beads were determined by Tandem-Mass-Tag mass spectrometry (TMT-MS). On-bead digestion, TMTpro 16plex labeling, alkylation/cysteine protection by iodoacetamide (IAA), stage tip desalting, and SpeedVac drying were performed following the manufacturer’s instructions and standard protocols (ThermoFisher, #A44520; Zhou, et al., 2024). Peptides were reconstituted in 0.1% formic acid for LC-MS analysis. TMT-labeling efficiency was verified to be more than 95%. Mass spectrometric data were collected on an Orbitrap Fusion Lumos instrument (using hrMS2-mode). Details of data acquisition and analysis were included in the Methods part of the original work.