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The possibility to record proteomes in high throughput and at high quality has opened new avenues for biomedical research, drug discovery, systems biology, and clinical translation. However, high-throughput proteomic experiments often require high sample amounts and can be less sensitive compared to conventional proteomic experiments. Here, we introduce and benchmark Zeno SWATH MS, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) to increase the sensitivity in high-throughput proteomic experiments. We demonstrate that when combined with fast micro- or analytical flow-rate chromatography, Zeno SWATH MS increases protein identification with low sample amounts. For instance, using 20 min micro-flow-rate chromatography, Zeno SWATH MS identified more than 5000 proteins consistently, and with a coefficient of variation of 6%, from a 62.5 ng load of human cell line tryptic digest. Using 5 min analytical flow-rate chromatography (800 µl/min), Zeno SWATH MS identified 4907 proteins from a triplicate injection of 2 µg of a human cell lysate, or more than 3000 proteins from a 250 ng tryptic digest. Zeno SWATH MS hence facilitates sensitive high-throughput proteomic experiments with low sample amounts, mitigating the current bottlenecks of high-throughput proteomics.

Mechanisms regulating the turnover of synaptic vesicle (SV) proteins are not well understood. They are thought to require poly-ubiquitination and degradation through proteasome, endo-lysosomal or autophagy-related pathways. Bassoon was shown to negatively regulate presynaptic autophagy in part by scaffolding Atg5. Here, we show that increased autophagy in Bassoon knockout neurons depends on poly-ubiquitination and that the loss of Bassoon leads to elevated levels of ubiquitinated synaptic proteins per se. Our data show that Bassoon knockout neurons have a smaller SV pool size and a higher turnover rate as indicated by a younger pool of SV2. The E3 ligase Parkin is required for increased autophagy in Bassoon-deficient neurons as the knockdown of Parkin normalized autophagy and SV protein levels and rescued impaired SV recycling. These data indicate that Bassoon is a key regulator of SV proteostasis and that Parkin is a key E3 ligase in the autophagy-mediated clearance of SV proteins.