Scalability is one of the crucial factors for single cell proteomics for elucidating heterogeneity in single cell and near single cell tissue input applications. Multiplexing or fast chromatography with low overhead time aids in upscaling proteome analyses. Here, we demonstrate applicability of Evosep Whisper Zoom for speeding up label free sample analysis up to 120 samples per day (SPD) and exceeding 1000SPD in a multiplexing approach on the timsTOF Ultra2.
HeLa cells were isolated at single cell level and 10 cell bulks into the proteoCHIP EVO96, directly lysed and digested using the cellenONE platform, transferred by centrifugation onto Evotips, separated in Whisper Zoom 120, 80 and 40SPD, and analyzed on a timsTOF Ultra2. Further, protein digests of HeLa, HEK293 and K562 cell lysates were labeled with a subset 16-plex TMTpro, combined to 9-plex at single cell equivalents (250 pg per label), and analyzed in Whisper Zoom 120 SPD.
Analyses of HeLa cell digest dilutions (Pierce) 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, 5000 proteins at 80SPD and 5500 protein groups at 40SPD speed and reaching 6500 protein groups at 120SPD, 7000 protein groups at 80SPD and 7700 protein groups at 40SPD from the higher loads. For 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.
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 and quantitative accuracy within and across TMT batches was observed, accurately separating HEK 293 samples from HeLa samples and K562 samples.