High-Field Asymmetric Waveform Ion Mobility Spectrometry: Practical Alternative for Cardiac Proteome Sample Processing

Ai, Lizhuo; Binek, Aleksandra; Kreimer, Simion; Ayres, Matthew; Stotland, Aleksandr; Eyk, Jennifer E. Van

doi:10.1021/acs.jproteome.3c00027

Cited by 3 publications

(1 citation statement)

References 42 publications

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“…Also of note is the number of identifications in heart tissue, which is a typically challenging for analysis due to the high-abundant sarcomeric myofilament proteins responsible for heart contractile motion that obscure low-abundant species, acting much like albumin in plasma. 25,26 When searched together with the other murine tissues, we identify over 10k protein groups corresponding to 8928 unique proteins. The ontology of these identifications is displayed in figure 4, with coverage of the Golgi apparatus, nucleus and mitochondria inset.…”

Section: Resultsmentioning

confidence: 99%

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

Hendricks,

Bhosale,

Keoseyan

et al. 2024

Preprint

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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.

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Section: Resultsmentioning

confidence: 99%

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

Hendricks,

Bhosale,

Keoseyan

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

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Liquid-phase separations coupled with ion mobility-mass spectrometry for next-generation biopharmaceutical analysis

Makey,

Ruotolo

2024

Expert Review of Proteomics

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Proteomics of the heart

Karpov,

Stotland,

Raedschelders

et al. 2024

Physiological Reviews

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Mass spectrometry-based proteomics is a sophisticated identification tool specializing in portraying protein dynamics at a molecular level. Proteomics provides biologists with a snapshot of context-dependent protein expression, isoform conformations, dynamic turnover information, and data on direct protein-protein interactions. Cardiac proteomics offers researchers and clinicians a deeper understanding of the molecular mechanisms that underscore cardiovascular disease, and is foundational to the development of future therapeutic interventions. This review encapsulates the evolution, current technologies, and future perspectives of proteomic-based mass spectrometry as it applies to the study of the heart. Key technological advancements have allowed for researchers to study proteomes at a single cell level, employ robot-assisted automation systems for enhanced sample preparation techniques, and the increase in fidelity of the mass spectrometers has allowed for the unambiguous identification of numerous dynamic post-translational modifications (PTMs). Animal models of cardiovascular disease, ranging from early animal experiments to current sophisticated models of heart failure with preserved ejection fraction (HFpEF), have provided the tools to study a challenging organ in the laboratory. Further technological development will pave way for the implementation of proteomics even closer within the clinical setting, allowing not only scientists but also patients to benefit from an understanding of protein interplay as it relates to cardiac disease physiology.

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High-Field Asymmetric Waveform Ion Mobility Spectrometry: Practical Alternative for Cardiac Proteome Sample Processing

Cited by 3 publications

References 42 publications

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

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

Liquid-phase separations coupled with ion mobility-mass spectrometry for next-generation biopharmaceutical analysis

Proteomics of the heart

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