An Inflection Point in High-Throughput Proteomics with Orbitrap Astral: Analysis of Biofluids, Cells, and Tissues

Abstract

This Technical Note presents a comprehensive proteomics workflow for the new combination of Orbitrap and Astral mass analyzers across biofluids, cells, and tissues. Central to our workflow is the integration of Adaptive Focused Acoustics (AFA) technology for cells and tissue lysis to ensure robust and reproducible sample preparation in a high-throughput manner. Furthermore, we automated the detergent-compatible single-pot, solid-phase-enhanced sample Preparation (SP3) method for protein digestion. The synergy of these advanced methodologies facilitates a robust and high-throughput approach for cell and tissue analysis, an important consideration in translational research. This work disseminates our platform workflow, analyzes the effectiveness, demonstrates the reproducibility of the results, and highlights the potential of these technologies in biomarker discovery and disease pathology. For cells and tissues (heart, liver, lung, and intestine) proteomics analysis by data-independent acquisition mode, identifications exceeding 10,000 proteins can be achieved with a 24 min active gradient. In 200 ng injections of HeLa digest across multiple gradients, an average of more than 80% of proteins have a CV less than 20%, and a 45 min run covers ∼90% of the expressed proteome. This complete workflow allows for large swaths of the proteome to be identified and is compatible with diverse sample types.

Keywords: Orbitrap Astral; PBMCs; automation; biomarker; high-throughput; mass spectrometry; missing proteins; plasma proteomics; tissue proteomics.

Figure 1

A) Comparison of the different gradients and DIA window sizes by %CV. Box plots are given for naive plasma (left) and HeLa (right) according to three different gradients, with two window sizes per gradient. Median %CVs are overlaid. B) CV plots of all identification in naïve (right) and depleted plasma (left), in both 24 (top) and 45 min (bottom) runs. Published FDA biomarkers are listed highlighted in red.

Figure 2

A) Protein abundance rank plots of the of naïve and depleted plasma samples in the 24 min 3 Th MS method. B) Proteins seen in antibody-depleted of top 14 abundant plasma proteins, PerCA precipitated, and Seer Proteograph plasma samples and their overlap with Naïve are shown in a Venn diagram.

Figure 3

Reactome pathways based on the molecular function of proteins identified in PBMC samples on the Astral 24 min runs. Node size represents the number of proteins and is filtered to display only pathways with 150 proteins or more. All nodes have significant adjusted p-value enrichment, with a range of 3.6e-3 to 9.9e-141.

Figure 4

Gene ontology of the identifications in murine heart tissue samples when coanalyzed with other murine tissues in DIA-NN. Coverage of Golgi, mitochondrial, and nuclear proteins within each section is further expanded.