Proteomic Profiling of Extracellular Vesicles Derived from Cerebrospinal Fluid of Alzheimer’s Disease Patients: A Pilot Study
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Selection
2.2. Separation of EVs from Human CSF Samples
2.3. Nanoparticle Tracking Analysis (NTA)
2.4. Transmission Electron Microscopy (TEM)
2.5. Protein Concentrations
2.6. In-Solution Digestion
2.7. Mass Spectrometry
2.7.1. Peptide Labeling with TMT 10-Plex Isobaric Labeling Kit
2.7.2. Nano-Liquid Chromatography and Tandem Mass-Spectrometry
2.7.3. Mass-Spectrometry Data Analysis
2.8. Measurement of Total Tau (t-tau) and Tau Phosphorylated on Threonine 231 (p-tau231)
2.9. Statistical Analysis
3. Results
3.1. Workflow for Protein Profiling of Extracellular Vesicles Separated from Human CSF
3.2. Biochemical and Morphological Characterization of EVs Separated from CSF
3.3. Comparison of AD, MCI, and CTRL CSF-Derived EV Proteins by TMT-Labeled Quantitative Proteomics Analysis
3.4. Molecules Altered during Progression to Alzheimer’s Disease from Mild Cognitive Impairment
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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For Label-Free Proteomics | AD | |||||
(n = 3) | ||||||
Age, mean | 82.7 ± 7.57 | |||||
Gender | 2M, 1F | |||||
For TMT-Label Proteomics | CTRL | MCI | AD | t-test a (p-value b) | t-test a (p-value b) | t-test a (p-value b) |
(n = 10) | (n = 10) | (n = 10) | (CTRL_MCI) | (CTRL_AD) | (MCI_AD) | |
Age, mean | 89.8 ± 6.12 | 89.0 ± 5.27 | 87.1 ± 4.41 | 0.313 (0.758) | 1.131 (0.273) | 0.874 (0.393) |
Gender c | 5M, 5F | 5M, 5F | 5M, 5F | |||
PMI, mean | 2.90 ± 0.85 | 2.94 ± 0.87 | 2.84 ± 0.49 | −0.106 (0.917) | 0.173 (0.865) | 0.299 (0.768) |
Braak stage | 2.8 ± 0.42 | 3.3 ± 0.67 | 5.4 ± 0.52 | −1.987 (0.062) | −12.333 (<0.001) | −7.814 (<0.001) |
Human CSF | EV Fraction | |
---|---|---|
Mode size (nm) a | 191 | 110 |
Particle number a,b | 4.6 × 109/500 μL CSF | 2.36 × 109/100 μL |
EV protein (μg) b,c | 1187.48 | 0.78 |
Particles/proteins (μg) | 3.87 × 106 | 3.03 × 109 |
Protein ID | Gene Symbol | log2 | log2 | log2 | One-Way ANOVA a | −log10 (p-Value | −log10 (p-Value | −log10 (p-Value |
---|---|---|---|---|---|---|---|---|
(AD/CTRL) | (MCI/CTRL) | (AD/MCI) | (AD_CTRL)) a | (MCI_CTRL)) | (AD_MCI)) | |||
P08107 | HSPA1A | 0.405 | −0.128 | 0.533 | 0.017 | 1.149 | 0.087 | 1.721 |
Q9P2B2 | PTGFRN | 0.882 | 0.391 | 0.491 | 0.024 | 1.721 | 0.268 | 0.75 |
P55786 | NPEPPS | 0.145 | −0.243 | 0.388 | 0.025 | 0.319 | 0.666 | 1.699 |
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Muraoka, S.; Jedrychowski, M.P.; Yanamandra, K.; Ikezu, S.; Gygi, S.P.; Ikezu, T. Proteomic Profiling of Extracellular Vesicles Derived from Cerebrospinal Fluid of Alzheimer’s Disease Patients: A Pilot Study. Cells 2020, 9, 1959. https://doi.org/10.3390/cells9091959
Muraoka S, Jedrychowski MP, Yanamandra K, Ikezu S, Gygi SP, Ikezu T. Proteomic Profiling of Extracellular Vesicles Derived from Cerebrospinal Fluid of Alzheimer’s Disease Patients: A Pilot Study. Cells. 2020; 9(9):1959. https://doi.org/10.3390/cells9091959
Chicago/Turabian StyleMuraoka, Satoshi, Mark P. Jedrychowski, Kiran Yanamandra, Seiko Ikezu, Steven P. Gygi, and Tsuneya Ikezu. 2020. "Proteomic Profiling of Extracellular Vesicles Derived from Cerebrospinal Fluid of Alzheimer’s Disease Patients: A Pilot Study" Cells 9, no. 9: 1959. https://doi.org/10.3390/cells9091959