Cross‐Platform Comparison of Chemoproteomics Workflows: Orbitrap‐TMT Versus timsTOF‐diaPASEF

ABSTRACT Chemoproteomics aims to achieve precise and comprehensive quantification of protein–small molecule interactions, yet methodological comparisons across quantitative proteomics workflows remain scarce. Here, we systematically benchmark tandem mass tag data‐dependent acquisition (TMT‐DDA) on Orbitrap Exploris/Eclipse instruments against label‐free data‐independent acquisition parallel accumulation–serial fragmentation (LFQ‐diaPASEF) on a timsTOF Pro 2 mass spectrometer when applied for 2‐dimensional thermal proteome profiling (2D‐TPP) and Kinobeads‐based chemoproteomics. Our findings demonstrate that TMT‐DDA provided more confident detection of compound‐induced thermal shifts despite having lower proteome coverage in high‐complexity 2D‐TPP datasets. In contrast, LFQ‐diaPASEF excelled in low‐complexity affinity enrichment experiments, achieving up to 63% broader total proteome coverage and comparable quantitative accuracy, even with ultra‐short gradients. Additionally, we show that normalization strategies in DIA require careful selection to avoid artifacts when transforming IC 50 s into apparent dissociation constants (K d app s). These findings emphasize that the trade‐offs inherent to each platform critically impact chemoproteomics experiments and that instrument–method pairings should be selected based on experimental complexity, quantitative precision, data completeness needs, and throughput demands.