Related Experiment Video
Updated: Mar 13, 2026
Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
Robust Label-free, Quantitative Profiling of Circulating Plasma Microparticle (MP) Associated Proteins
Sophie Braga-Lagache1, Natasha Buchs1, Mircea-Ioan Iacovache2
1From the ‡Department of Clinical Research.
Abstract:
Cells of the vascular system release spherical vesicles, called microparticles, in the size range of 0.1-1 μm induced by a variety of stress factors resulting in variable concentrations between health and disease. Furthermore, microparticles have intercellular communication/signaling properties and interfere with inflammation and coagulation pathways. Today's most used analytical technology for microparticle characterization, flow cytometry, is lacking sensitivity and specificity, which might have led to the publication of contradicting results in the past.We propose the use of nano-liquid chromatography two-stage mass spectrometry as a nonbiased tool for quantitative MP proteome analysis.For this, we developed an improved microparticle isolation protocol and quantified the microparticle protein composition of twelve healthy volunteers with a label-free, data-dependent and independent proteomics approach on a quadrupole orbitrap instrument.Using aliquots of 250 μl platelet-free plasma from one individual donor, we achieved excellent reproducibility with an interassay coefficient of variation of 2.7 ± 1.7% (mean ± 1 standard deviation) on individual peptide intensities across 27 acquisitions performed over a period of 3.5 months. We show that the microparticle proteome between twelve healthy volunteers were remarkably similar, and that it is clearly distinguishable from whole cell and platelet lysates. We propose the use of the proteome profile shown in this work as a quality criterion for microparticle purity in proteomics studies. Furthermore, one freeze thaw cycle damaged the microparticle integrity, articulated by a loss of cytoplasm proteins, encompassing a specific set of proteins involved in regulating dynamic structures of the cytoskeleton, and thrombin activation leading to MP clotting. On the other hand, plasma membrane protein composition was unaffected. Finally, we show that multiplexed data-independent acquisition can be used for relative quantification of target proteins using Skyline software. Mass spectrometry data are available via ProteomeXchange (identifier PXD003935) and panoramaweb.org (https://panoramaweb.org/labkey/N1OHMk.url).
Insights
Vascular microparticles (MPs) are key in cell signaling. This study introduces nano-liquid chromatography-mass spectrometry for precise MP proteome analysis, establishing a purity standard and revealing damage from freeze-thaw cycles.
Area of Science:
- Biochemistry
- Cell Biology
- Proteomics
Background:
- Vascular cells release microparticles (MPs), 0.1-1 μm vesicles involved in intercellular communication, inflammation, and coagulation.
- Current analytical methods like flow cytometry lack sensitivity and specificity for MP characterization, leading to inconsistent research findings.
- Accurate quantitative analysis of the MP proteome is crucial for understanding their role in health and disease.
Purpose of the Study:
- To establish nano-liquid chromatography two-stage mass spectrometry (nLC-MS/MS) as a nonbiased tool for quantitative microparticle proteome analysis.
- To develop an improved microparticle isolation protocol for enhanced purity and reproducibility.
- To characterize the proteome profile of microparticles from healthy volunteers and assess MP integrity under stress conditions.
Main Methods:
- Developed an improved microparticle isolation protocol.
- Utilized label-free, data-dependent, and data-independent proteomics approaches with nLC-MS/MS on a quadrupole orbitrap instrument.
- Analyzed platelet-free plasma from twelve healthy volunteers, with extensive reproducibility assessments.
Main Results:
- Achieved high reproducibility (CV 2.7 ± 1.7%) in peptide intensity measurements across multiple acquisitions.
- Demonstrated that the microparticle proteome is highly consistent among healthy individuals and distinct from whole cell and platelet lysates.
- Identified that freeze-thaw cycles damage MP integrity, causing loss of cytoplasmic proteins and thrombin activation, while plasma membrane proteins remain unaffected.
Conclusions:
- The established nLC-MS/MS method provides a robust and reproducible platform for quantitative microparticle proteome analysis.
- The proteome profile serves as a quality criterion for microparticle purity in proteomics studies.
- Understanding MP integrity and damage mechanisms is vital for reliable research and clinical applications.
