(1378-C) Synergies in automated sample preparation and short gradient LC-MS acquisition advances high-throughput discovery proteomics.
Tuesday, February 6, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: Mass spectrometry-based proteomics enables comprehensive and precise protein identification and quantification across large sample cohorts. Labor-intensive sample preparation workflows often limit throughput of large-scale studies and reduce the statistical power by introducing technical variability. Additionally, to fully capture the high complexity of biological samples, long high-performance liquid chromatography (HPLC) gradients are necessary, which limits the number of measured samples per day on a mass spectrometer (MS). Higher sample numbers are needed in state of the art in biological studies, driven by dynamic biological systems and the need for statistical power. This emphasizes the need for cost-effective high-throughput proteomics workflows. Here we present a fully automated sample preparation workflow for proteomics that independently processes samples from biological input to MS-ready peptide output. The fully integrated automated liquid handling platform enables comprehensive sample preparation without the need for human interaction. The workflow is designed for the simultaneous processing of two 96-well plates in a single run and offers flexibility in processing different sample types, such as cultured cells, lysed tissue or biofluids. By combining this system with the timsTOF high-throughput (HT) MS, coverage and sensitivity in proteome analysis can be improved thanks to TIMS (Trapped Ion Mobility Spectrometry) and PASEF (Parallel Accumulation-Serial Fragmentation) technology. Its high-throughput design enables rapid, accurate, and efficient processing of a large number of samples, making it a versatile choice for large-scale proteomics studies. To demonstrate the synergy of automated sample-preparation in conjunction with short gradient LC-MS analysis we processed ten distinct human cell lines in triplicates. Within only 31h total duration from the start of sample preparation to end of the MS acquisitions . We identified 9,547 protein groups on average per cell line. We calculated a median coefficient of variation (CV) of 7.9% on protein level, that demonstrates a high degree of standardization and overall reduction of technical variability compared to our internal standard semi-automated protocol. The high quantitative precision achieved allows for robust biological insights, as demonstrated by the impressive data completeness of 97.3% at the protein level. In addition, 87% of the quantified protein groups had a CV below 20%, confirming the precision and reproducibility of our quantitative analysis. The total 11,789 proteins identified across all samples resulted in a clearly distinguishable separation according to their origins in the principal component analysis. This enables a deeper exploration of the cellular proteome than ever before, achieved at a rapid pace. The implemented workflow drastically increases laboratory efficiency by reducing human input by more than 90%, while maintaining sample output quality and achieving better reproducibility across preparations over time. Through the use of short gradients on the timsTOF HT, large-scale studies become feasible, significantly amplifying the throughput of proteomic analyses. In summary, the combination of fully automated proteomics sample preparation with the rapid timsTOF acquisition method enables in-depth exploration of the cellular proteome with remarkable speed and depth.