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

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.

Blood to Biomarker Quantitation in Under One Hour with Rapid Proteomics using a Hyperthermoacidic Protease.

Multi-step multi-hour tryptic proteolysis has limited the utility of bottom-up proteomics for cases that require immediate quantitative information. The recently available hyperthermoacidic (HTA) protease "Krakatoa" digests samples in a single 5 to 30-minute step at pH 3 and >80 °C; conditions that disrupt most cells and tissues, denature proteins, and block disulfide reformation. The combination of quick single-step sample preparation with high throughput dual trapping column single analytical column (DTSC) liquid chromatography-mass spectrometry (LC-MS) achieves "Rapid Proteomics" in which the time from sample collection to actionable data is less than 1 hour. The presented development and systematic evaluation of this methodology found reproducible quantitation of over 160 proteins from just 1 microliter of whole blood. Furthermore, the preference of the HTA-protease for intact proteins over peptides allows for sensitive targeted quantitation of the Angiotensin I and II bioactive peptides in under half an hour. With these methods we analyzed serum and plasma from 53 individuals and quantified Angiotensin and proteins that were not detected with trypsin. This assessment of Rapid Proteomics suggests that concentration of circulating protein and peptide biomarkers could be measured in almost real-time by LC-MS. TOC Figure: Rapid proteomics enables near real-time monitoring of circulating blood biomarkers. One microliter of blood is collected every 8 minutes, digested for 20 minutes, and then analyzed by targeted mass spectrometry for 8 minutes. This results in a 30-minute delay with datapoints every 8 minutes.

An inflection point in high-throughput proteomics with Orbitrap Astral: analysis of biofluids, cells, and tissues.

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, a technique that streamlines the process by combining purification and digestion steps, thereby reducing sample loss and improving efficiency. The synergy of these advanced methodologies facilitates a robust and high-throughput approach for cells and tissue analysis, an important consideration in translational research. This work disseminates our platform workflow, analyzes the effectiveness, demonstrates 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-minute active gradient. In 200ng injections of HeLa digest across multiple gradients, an average of more than 80% of proteins have a CV less than 20%, and a 45-minute run covers ~90% of the expressed proteome. In plasma samples including naive, depleted, perchloric acid precipitated, and Seer nanoparticle captured, all with a 24-minute gradient length, we identified 87, 108, 96 and 137 out of 216 FDA approved circulating protein biomarkers, respectively. This complete workflow allows for large swaths of the proteome to be identified and is compatible across diverse sample types.