Thin-diaPASEF: diaPASEF for maximizing proteome coverage in single-shot proteomics

Abstract

Proteomics using mass spectrometry (MS) has significantly advanced, offering deep insights into complex proteomes. The timsTOF MS platform with its parallel accumulation-serial fragmentation (PASEF) technology has achieved high scan speeds and high-quality spectra. Bukers timsTOF HT, which features TIMS-XR technology, offers an improved dynamic range and analysis depth, supporting high sample loadings. Moreover, various improvements to the data-independent acquisition method based on the PASEF technology (diaPASEF) have been reported. Despite these advancements, most high-level deep proteomic reports are based on the Orbitrap Astral and Orbitrap Exploris 480, and analytical systems using timsTOF MS still require improvement. Here, Brukers timsTOF HT was used to validate and optimize key diaPASEF parameters, leading to the development of a Thin-diaPASEF method. This method provides a high quantitative accuracy and consistency. In our validation, 9,400 proteins were identified in a single shot from HEK cells (strictly controlled Protein false discovery rate < 1%), the highest number analyzed by the timsTOF MS series using standard human cultured cells. Furthermore, by combining Thin-diaPASEF with an improved Lycopersicon esculentum lectin method, over 5,000 proteins were identified in a 24-sample/d analysis, and we succeeded in constructing a system with high proteome coverage that can be used for biomarker discovery.