A high-throughput, in-depth investigation into the immunological effects of Lipopolysaccharide challenged mice in neat plasma on Orbitrap astral mass spectrometer

Lipopolysaccharide (LPS) inhalation stimulates an immune response in mice and serves as a preclinical model for investigating potential therapeutics for inflammatory related disorders such as acute bronchitis. Here mice were treated and challenged with LPS and compared to healthy, unchallenged mice to gain insights into immunological response and identify key down regulated and upregulated proteins of importance. Traditionally, neat plasma requires long runtimes of over 1hr to achieve the depth of coverage needed for meaningful insights by mass spectrometry. Here the Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer was optimized and assessed for high-throughput analysis while still maintaining depth of coverage.

Neat plasma from five biological replicates of LPS challenged and unchallenged mice were prepared by automated sample preparation on the Thermo Scientific™ AccelerOme™ with LysC/trypsin digestion that yielded peptides ready for separation by Thermo Scientific™ Vanquish™Neo™ and detection by Orbitrap Astral™ mass spectrometer. Separation was performed by reverse phase gradient with Ionopticks columns for various sample throughputs of 20SPD, 60SPD, and 180SPD to compare depth of coverage and quantitative accuracy. Three technical replicates were acquired to assess instrument reproducibility. A gas phase fractionation library was created for the 60SPD method and used to search the data for maximum protein identification. Data collected was analyzed by Thermo Scientific™ Proteome Discoverer™ 3.1, DIANN, and Spectronaut^®18 software for comparison. Over 3600 protein groups and 25400 peptides were detected with the 20SPD method searched with DIANN with no library applied. This was over 40% and 86% improvement over the 60SPD and 180SPD method respectively.

Results showed over 93% quantifiable IDs and median coefficient of variance (CV) below 10% for technical replicates for different run times. The gas phase fractionation library for 60SPD was used to process results and yielded a 44% improvement from 1680 protein IDs to 2427 protein IDs. The five biological replicates for the LPS challenged mice versus the unchallenged mice yielded 2169 and 1914 protein IDs respectively and were reproducible and robust across all five biological replicates. The LPS challenged results for the 60SPD method were 76% quantifiable in all five replicates and 91% quantifiable in three out of five replicates. Proteins were ranked on a ranked protein list and spanned nearly six order of magnitude and were clustered on a PCA plot to show the differences observed between the two treated and untreated groups. Next, differential expression was performed with a p-value <0.001 and +/- 2 Log2FC as cut-off, revealing 35 upregulated and two downregulated proteins in LPS challenged plasma versus unchallenged plasma. Orbitrap Astral mass spectrometer optimized for high-throughput and depth of coverage yielded >3600 protein groups in LPS challenged neat plasma.