Evaluation of Deep and Dynamic Proteomic Screening Strategies at Sub-50 Hz Scan Speed and without Robotic Sample Preparation

Abstract Advances in ultrafast mass analyzer technology and procedural automation have enabled proteomic screening at the throughput of hundreds of proteomes per day. However, these approaches often require expensive instrumentation upgrades and robotic automation that remain inaccessible to many research laboratories and core facilities. In this study, we address the feasibility of scaling up proteomic screening capabilities with minimal upgrade cost by focusing on (a) strategies for nonautomated high-throughput sample preparation from 96-well cell culture, (b) data acquisition on sub-50 Hz scan speed hybrid and tribrid Orbitrap instruments, and (c) data analysis strategies for label-free and labeled proteomic screening. We find that the 96-well format STrap, in combination with C18 plates, provides the most robust throughput for a nonautomated sample preparation workflow. Furthermore, we show that for static proteomes, an isobaric tandem mass tag (TMT)-based multiplexing approach provides deeper and more precise proteome coverage, whereas data-independent acquisition (DIA) is more accurate, albeit with a reduced dynamic range and more missing values. Finally, we extend the optimized workflow to proteome turnover studies using pulsed stable isotope labeling by amino acids in cell culture (pSILAC), highlighting the key advantages and trade-offs of DIA and TMT data-dependent acquisition strategies for capturing protein translation.