Phosphoproteomic Analysis of Breast Cancer-Derived Small Extracellular Vesicles Reveals Disease-Specific Phosphorylated Enzymes
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culturing and sEV Isolation
2.2. Differential Ultracentrifugation (UC)
2.3. Quantification of sEVs by Nanoparticle Tracking Analysis (NTA)
2.4. Sample Preparation for Phosphoproteomics
2.5. Phosphopeptide Enrichment by IMAC and TiO2
2.6. Nano-LC-MS/MS
2.7. MS Spectra Processing
2.8. Data Filtering and Phosphorylation Site Localization
2.9. Disease and Functional Annotation Analysis
2.10. Data Availability
2.11. ATP-Citrate Synthase Activity Assay
2.12. Phosphofructokinase Activity Assay
2.13. SIRT1 and SIRT6 Activity Assay
2.14. Western Blot
3. Results
3.1. Isolation of sEV
3.2. Overall Phosphoproteome Profiling
3.3. Phosphorylation Site Distributions, Phosphorylation Motifs and Predicted Potential Kinases for Identified Phosphorylation Sites
3.4. Identified Phosphoproteins in the Context of Cancer/Breast Cancer
3.5. Functional and Pathway Analysis of Identified Phosphoproteins
3.6. Analysis of ACLY, PKFM, SIRT1, and SIRT6 in Cells and Their sEVs
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Enzyme | Gene | Number of Phosphorpeptide Identifications | MCF7 sEV | MDA-MB-231 sEV | ||
---|---|---|---|---|---|---|
CID | HCD | CID | HCD | |||
Found in sample replicates (Number/3) | ||||||
ATP citrate lyase | ACLY | 8 | 3 | 3 | 1 | 1 |
6-Phosphofructokinase | PFKM | 8 | 1 | 0 | 3 | 3 |
Sirtuin 1 | SIRT1 | 7 | 3 | 1 | 0 | 3 |
CTP synthetase 1 | CTPS1 | 7 | 1 | 0 | 3 | 3 |
Sirtuin 6 | SIRT6 | 6 | 0 | 3 | 0 | 3 |
Acetyl-CoA synthetase 2 | ACSS2 | 5 | 2 | 3 | 0 | 0 |
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Minic, Z.; Hüttmann, N.; Poolsup, S.; Li, Y.; Susevski, V.; Zaripov, E.; Berezovski, M.V. Phosphoproteomic Analysis of Breast Cancer-Derived Small Extracellular Vesicles Reveals Disease-Specific Phosphorylated Enzymes. Biomedicines 2022, 10, 408. https://doi.org/10.3390/biomedicines10020408
Minic Z, Hüttmann N, Poolsup S, Li Y, Susevski V, Zaripov E, Berezovski MV. Phosphoproteomic Analysis of Breast Cancer-Derived Small Extracellular Vesicles Reveals Disease-Specific Phosphorylated Enzymes. Biomedicines. 2022; 10(2):408. https://doi.org/10.3390/biomedicines10020408
Chicago/Turabian StyleMinic, Zoran, Nico Hüttmann, Suttinee Poolsup, Yingxi Li, Vanessa Susevski, Emil Zaripov, and Maxim V. Berezovski. 2022. "Phosphoproteomic Analysis of Breast Cancer-Derived Small Extracellular Vesicles Reveals Disease-Specific Phosphorylated Enzymes" Biomedicines 10, no. 2: 408. https://doi.org/10.3390/biomedicines10020408
APA StyleMinic, Z., Hüttmann, N., Poolsup, S., Li, Y., Susevski, V., Zaripov, E., & Berezovski, M. V. (2022). Phosphoproteomic Analysis of Breast Cancer-Derived Small Extracellular Vesicles Reveals Disease-Specific Phosphorylated Enzymes. Biomedicines, 10(2), 408. https://doi.org/10.3390/biomedicines10020408