Scalable single cell analysis towards 1000 samples per day using the Evosep whisper zoom methods on the timsTOF Ultra2

Scalability is one of the crucial factors for single cell proteomics to allow meaningful data collection with reliable and robust statistical power for elucidating heterogeneity in ultra-high sensitive single cell and near single cell tissue input applications. Multiplexing as well as fast chromatography with low overhead time aids in upscaling proteome analyses. Here, we demonstrate applicability of the new Whisper Zoom methods on the Evosep system for speeding up label free sample analysis up to 120 samples per day (SPD) and exceeding >1000 samples per day in a multiplexing approach with data acquisition on the timsTOF Ultra2.

HeLa and HEK293 as well as small HEK 293 spheroids were isolated into the proteoCHIP® EVO 96, directly lysed and digested using the cellenONE platform. Samples were transferred by centrifugation onto Evotips, separated in Whisper Zoom 120, 80 and 40SPD, analyzed on timsTOF Ultra2 in dia-PASEF® mode and processed with Spectronaut 19 using directDIA+. Further, protein digests were labelled with a subset 16-plex TMTpro, combined to 9-plex single cell equivalent samples (250 pg per labeled), analyzed in Whisper Zoom 120SPD on the timsTOF Ultra2 in dda-PASEF and analyzed with Spectromine 4.5.

Analyses of HeLa cell digest dilutions from 50 ng to 0.25 ng were performed to assess sensitivity of the different Whisper Zoom methods identifying more than 4000 protein groups from 250 pg at 120SPD, 5,000 proteins at 80SPD and 5500 protein groups at 40SPD speed and reaching 6,500 protein groups at 120SPD, 7000 protein groups at 80SPD and 7700 protein groups at 40SPD from the higher loads. From single Hela cells, protein identification rates correlated with gradient length, identifying on average 2500 protein groups at 120SPD, 3000 protein groups at 80SPD and 4000 protein groups at 40SPD. This setup was then used to investigate cellular responses to LPS treatment on single cell level (HEK 293 cells 40x treated, 40x controls) and small spheroids (3 – 10 cells per spheroid; 12x treated, 12x controls). Results were compared to responses seen in 30 cell isolation bulk samples (4x treated, 4x controls). As expected, sample to sample variability decreased with an increasing cell number. However, comparing protein groups identified in the undisturbed non-treated samples, 65% of all proteins identified in 30 cell bulk were identified at single cell level and increased to 85% on small spheroid level. LPS treatment responses on 30 cell bulk samples, spheroids and single cell level were comparable showing expected activation of inflammatory pathways.

The 9-plex multiplexing approach run at Whisper Zoom 120SPD speed resulted across the 1080 samples on average in the identification of 1500 protein group per label/cell type. Excellent tip to tip chromatographic reproducibility as well as quantitative accuracy within and across TMT batches was observed, accurately separating the 360 HEK 293 samples from the 360 HeLa samples and the 360 K562 samples.