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

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 IC₅₀s into apparent dissociation constants (K_d ^apps). 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.