Advancing Target Identification of Nitrated Phospholipids in Biological Systems by HCD Specific Fragmentation Fingerprinting in Orbitrap Platforms
Abstract
:1. Introduction
2. Results
2.1. Optimization of the Normalized Collision Energy for the Study of Nitrated PL Standards
2.2. Identification of Nitrated PL in Cell Lipid Extracts
3. Discussion
4. Materials and Methods
4.1. Reagents/Chemicals
4.2. Nitration of Phospholipids Using In Vitro Model Systems
4.3. Phospholipid Quantification by Phosphorous Measurement Assay
4.4. HCD-Orbitrap Instrumental Conditions
4.5. Cell Culture and Treatments of Adrenal Carcinoma SW13/cl.2 Cells
4.6. Lipid Extraction from SW13/cl.2 Cells
4.7. HPLC-ESI-MS and MS/MS Analysis
4.8. Data and Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample Availability: Samples of the compounds are not available from the authors. |
Typical Neutral Losses and Product Ions Observed for NO2-PC and NO2-PE in Positive Ion Mode | |||||||
Neutral Losses | Proposed Identification | (POPC + NO2 + H)+ | (POPE + NO2 + H)+ | (PLPC + NO2 + H)+ | (PLPE + NO2 + H)+ | (PAPC + NO2 + H)+ | (PAPE + NO2 + H)+ |
805.8 | 763.6 | 803.6 | 761.5 | 827.8 | 785.6 | ||
18 Da | -H2O | 787.6 | 745.5 | 785.5 | 743.5 | 809.6 | 767.5 |
43 Da | -C2H5N | --- | 720.4 | -- | 718.4 | -- | 742.5 |
47 Da | -HNO2 | 758.7 | 716.5 | 756.5 | 714.6 | 780.6 | 738.5 |
59 Da | -C3H9N | 746.5 | -- | 744.34 | -- | 768.7 | -- |
90 Da (43 + 47) | -(C2H5N + HNO2) | -- | 673.4 | -- | 671.6 | -- | 695.5 |
106 Da (59 + 47) | -(C3H9N + HNO2) | 699.6 | --- | 697.4 | -- | 721.5 | -- |
141 Da | -C2H8PO4N | -- | 622.5 | -- | 620.5 | -- | 644.5 |
183 Da | -C5H14PO4N | 622.6 | -- | 620.6 | -- | 644.6 | -- |
188 Da (141 + 47) | -(C2H8PO4N + HNO2) | --- | 575.4 | -- | 573.4 | -- | 597.6 |
230 Da (183 + 47) | -(C5H14PO4N + HNO2) | 575.5 | -- | 573.6 | -- | 597.7 | -- |
238 Da | -R1C=C=O | 567.6 | 525.4 | 565.4 | 523.3 | 589.3 | 547.4 |
256 Da | -R1COOH | 549.6 | 507.4 | 547.4 | 505.5 | 571.5 | 529.5 |
Product Ions | |||||||
Nitro Oleic Acid (NO2-OA) | Nitro Linoleic Acid (NO2-LA) | Nitro Arachidonic Acid (NO2-AA) | |||||
(NO2-FA + H)+ | 328.3 | 328.3 | 326.4 | 326.4 | 350.3 | 350.3 | |
(NO2-FA-H2O + H)+ | 310.3 | 310.3 | 308.3 | 308.3 | 332.3 | 332.3 | |
(NO2-FA-2H2O + H)+ | 292.3 | 292.3 | 290.3 | 290.3 | 314.2 | 314.2 | |
(NO2-FA-NO2 + H)+ | 281.3 | 281.3 | 279.3 | 279.3 | 303.3 | 303.3 |
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Neves, B.; Duarte, S.; Domingues, P.; Pérez-Sala, D.; Oliveira, M.M.; Domingues, M.d.R.; Melo, T. Advancing Target Identification of Nitrated Phospholipids in Biological Systems by HCD Specific Fragmentation Fingerprinting in Orbitrap Platforms. Molecules 2020, 25, 2120. https://doi.org/10.3390/molecules25092120
Neves B, Duarte S, Domingues P, Pérez-Sala D, Oliveira MM, Domingues MdR, Melo T. Advancing Target Identification of Nitrated Phospholipids in Biological Systems by HCD Specific Fragmentation Fingerprinting in Orbitrap Platforms. Molecules. 2020; 25(9):2120. https://doi.org/10.3390/molecules25092120
Chicago/Turabian StyleNeves, Bruna, Sofia Duarte, Pedro Domingues, Dolores Pérez-Sala, Maria Manuel Oliveira, Maria do Rosário Domingues, and Tânia Melo. 2020. "Advancing Target Identification of Nitrated Phospholipids in Biological Systems by HCD Specific Fragmentation Fingerprinting in Orbitrap Platforms" Molecules 25, no. 9: 2120. https://doi.org/10.3390/molecules25092120