Optimizing data-independent acquisition for the Orbitrap exploris 240: enhanced proteome coverage and quantitative accuracy and precision

Proteomic analysis through data-independent acquisition (DIA) has advanced significantly, driven by improvements in instrumentation, data analysis, and acquisition strategies. These advancements have enabled deep proteome coverage and enhanced quantitative accuracy and precision. However, achieving such high metrics requires careful optimization of liquid chromatography (LC) and mass spectrometry (MS) methods.

In this study, we optimized DIA parameters for the new Orbitrap Exploris 240 and evaluated the performance of various acquisition strategies. We defined the LC elution peak width using a 124-minute gradient, from 7 % to 45 % of acetonitrile concentration, in a 50-cm analytical column and optimized MS parameters, including MS resolution, isolation width, and m/z range, to achieve a median of at least 10 points per peak. Additionally, we tested dynamic and automatic injection times (IT) and various MS2 AGCs in the SWATH-MS acquisition strategy, followed by identification and quantification tests using different HEK cell lysate peptide amounts. Further, library generation with gas phase fractionation (GPF) and staggered window approaches were evaluated, and the optimized methods' quantitative merits were assessed using a complex mixture of proteomes from three species with known fold change (FC) values. Identification and quantification were performed in Spectronaut with default parameters.

Initial tests achieved a median LC peak width of 17 seconds, suggesting that an isolation window of 16 m/z with 15k MS2 resolution in the 400-1000 m/z range resulted in a median of 10 points per peak, suitable for quantitation. Dynamic IT did not significantly improve the identification rate compared to auto mode, nor did varying MS2 AGC between 300% and 800%. Injecting peptide amounts from 0.14 µg to 1 µg resulted in identifying 5311 to 6879 protein groups (PGs), with a plateau near 0.7 µg.

The spectral library created from GPF identified 2495 more precursors and 697 more PGs compared to a library-free strategy. The staggered window approach for GPF further increased precursor identifications by 2.36% compared to SWATH-MS, although it did not yield significant quantitative differences.

Our optimized methods identified over 8000 protein groups from human HEK293 cells, achieving a median coefficient of variation (CV) of 5.0%. The complex proteome mixture analysis showed absolute median deviations of 0.51 and 0.13 from expected FC values of 2 and 0.5, respectively. These results demonstrate a deep proteome coverage, quantitative accuracy, and precision achievable with the optimized methods for the Exploris 240 mass spectrometer.