Strategy for Microscale Extraction and Proteome Profiling of Peripheral Blood Mononuclear Cells

Li, Yang; Weng, Shuang; Qian, Xiaohong; Wang, Mingchao; Ying, Wantao

doi:10.1021/acs.analchem.1c05365

Cited by 3 publications

(2 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Studying PBMCs provides a detailed understanding of how specific cells respond to various stimuli such as infections, cancers, or therapeutic drugs. 23,24 To the best of our knowledge, close to 9000 protein identification from PBMCs represents the most extensive proteome coverage achieved in a 24-minute analysis. Figure 3 shows highly significant enrichment of reactome pathways seen in PBMCs covering metabolism, disease, cell cycle, and immune response, reflecting the variety of responses to stimuli that could be interrogated in a 24-minute analysis.…”

Section: Gradientmentioning

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

View full text Add to dashboard Cite

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.

show abstract

Section: Gradientmentioning

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

View full text Add to dashboard Cite

show abstract

“…They are responsible for activating and regulating immune responses to pathogens and are frequently targeted for numerous diagnostic, therapeutic, and research purposes. Separating these cells from whole blood is often a first step towards performing complex analyses and treatments such as measuring cytokine release 1 – 3 , genome sequencing 4 – 6 , and cell therapies 7 , 8 . Due to their large and versatile area of usage, separating PBMCs from patient blood is common in many laboratories.…”

Section: Introductionmentioning

confidence: 99%

Label-free separation of peripheral blood mononuclear cells from whole blood by gradient acoustic focusing

Alsved,

Rezayati Charan,

Ohlsson

et al. 2024

Sci Rep

View full text Add to dashboard Cite

Efficient techniques for separating target cells from undiluted blood are necessary for various diagnostic and research applications. This paper presents acoustic focusing in dense media containing iodixanol to purify peripheral blood mononuclear cells (PBMCs) from whole blood in a label-free and flow-through format. If the blood is laminated or mixed with iodixanol solutions while passing through the resonant microchannel, all the components (fluids and cells) rearrange according to their acoustic impedances. Red blood cells (RBCs) have higher effective acoustic impedance than PBMCs. Therefore, they relocate to the pressure node despite the dense medium, while PBMCs stay near the channel walls due to their negative contrast factor relative to their surrounding medium. By modifying the medium and thus tuning the contrast factor of the cells, we enriched PBMCs relative to RBCs by a factor of 3600 to 11,000 and with a separation efficiency of 85%. That level of RBC depletion is higher than most other microfluidic methods and similar to that of density gradient centrifugation. The current acoustophoretic chip runs up to 20 µl/min undiluted whole blood and can be integrated with downstream analysis.

show abstract

A micro-costing study of mass-spectrometry based quantitative proteomics testing applied to the diagnostic pipeline of mitochondrial and other rare disorders

Santos Gonzalez,

Hock,

Thorburn

et al. 2024

Orphanet J Rare Dis

View full text Add to dashboard Cite

Background Mass spectrometry-based quantitative proteomics has a demonstrated utility in increasing the diagnostic yield of mitochondrial disorders (MDs) and other rare diseases. However, for this technology to be widely adopted in routine clinical practice, it is crucial to accurately estimate delivery costs. Resource use and unit costs required to undertake a proteomics test were measured and categorized into consumables, equipment, and labor. Unit costs were aggregated to obtain a total cost per patient, reported in 2023 Australian dollars (AUD). Probabilistic and deterministic sensitivity analysis were conducted to evaluate parameter uncertainty and identify key cost drivers. Results The mean cost of a proteomics test was $897 (US$ 607) per patient (95% CI: $734-$1,111). Labor comprised 53% of the total costs. At $342 (US$ 228) per patient, liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) was the most expensive non-salary component. An integrated analysis pipeline where all the standard analysis are performed automatically, as well as discounts or subsidized LC-MS/MS equipment or consumables can lower the cost per test. Conclusions Proteomics testing provide a lower-cost option and wider application compared to respiratory chain enzymology for mitochondrial disorders and potentially other functional assays in Australia. Our analysis suggests that streamlining and automating workflows can reduce labor costs. Using PBMC samples may be a cheaper and more efficient alternative to generating fibroblasts, although their use has not been extensively tested yet. Use of fibroblasts could potentially lower costs when fibroblasts are already available by avoiding the expense of isolating PBMCs. A joint evaluation of the health and economic implications of proteomics is now needed to support its introduction to routine clinical care.

show abstract

Strategy for Microscale Extraction and Proteome Profiling of Peripheral Blood Mononuclear Cells

Cited by 3 publications

References 21 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

Label-free separation of peripheral blood mononuclear cells from whole blood by gradient acoustic focusing

A micro-costing study of mass-spectrometry based quantitative proteomics testing applied to the diagnostic pipeline of mitochondrial and other rare disorders

Contact Info

Product

Resources

About