Plasma Proteomics Across Three Generations of Mass Spectrometry Instruments: Lessons for Biofluid Method Optimisation

In biomarker discovery phases clinical proteomics provides large-scale identification and quantification of proteins in donor samples. For these studies plasma is the most frequently used biofluid, as it reflects both physiological and pathological states of the circulating proteome. However, the vast dynamic range of proteins in plasma remains a significant challenge, where low-abundance biomarkers are suppressed by high-abundance proteins, despite substantial technological improvements. Often the focus of improving outcomes in plasma proteomic workflows are within the biofluid's sample preparation, although adjusting MS methods dramatically improves detection and sampling of lower abundance proteins. Herein we have benchmarked various liquid chromatography (LC) and data-independent acquisition (DIA) methods across three generations of mass spectrometry instruments: timsTOF Pro (2017), Orbitrap Eclipse (2020), and Orbitrap Astral (2023). This study explored 27 combinations of LC-MS methods across 200 hours of instrument acquisition time, encompassing varying LC and MS settings. While each instrument generation significantly improved performance, each instrument also revealed a unique and different tuneable range to improve performance in plasma samples, highlighting the benefit of investing in plasma-specific method development for any mass spectrometer. We also evaluated the detection and quantification capabilities of each instrument via a unique approach of mixing paired platelet poor plasma (PPP) and platelet rich plasma (PRP), introducing linear contamination markers for quantitative assessment. This approach tested each instrument's sensitivity to detect low-abundance peptides and evaluated their quantitative accuracy. Lastly, we performed testing of the Orbitrap Astral's parallel ion processing capabilities with the aim of improving low-abundance protein identifications using gas-phase enrichment (GPE). More specifically, we show that optimisation of MS2 AGC targets and injection time enhanced GPE of low-abundance peptides, improving detection in plasma samples.