Epitomics: Analysis of Plasma C9 Epitope Heterogeneity in the Plasma of Lung Cancer Patients and Control Subjects
Abstract
:1. Introduction
2. Results
2.1. Statistical Validation of C9 PEP Data
2.2. Validation of Monoclonal Antibodies
2.3. Investigation of C9 Epitopes
2.4. C9 Epitope and Proteoform Heterogeneity in Control and Lung Cancer Subjects
3. Discussion
4. Materials and Method
4.1. Pooled Plasma Samples
4.2. C9 Immunoprecipitation
4.3. SDS-PAGE
4.4. Staining
4.5. Western Blotting
4.6. Mass Spectrometry Analysis
4.6.1. Sample Preparation
4.6.2. Mass Spectrometry
4.6.3. Data Interpretation
4.7. mAb Biotinylation
4.8. C9 Epitope Test
4.9. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Sample ID | Position 415 gly C9 Normalized Intensity to the Most Intense Unmodified C9 Peptide | Position 415 gly C9 Ctrl/LC Ratio |
---|---|---|
BSI0449 Ctrl | 0.67 | 1.25 |
BSI0449 LC | 0.54 | |
BSI0581 Ctrl | 1.02 | 4.88 |
BSI0581 LC | 0.21 | |
BSI0639 Ctrl | 1.00 | 5.83 |
BSI0639 LC | 0.17 |
Samples ID | C9 | C4A | HRNR | HSP90AB1 | HSPA8 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Num Unique | Peptide Count | Num Unique | Peptide Count | Coverage % | Num Unique | Peptide Count | Coverage % | Num Unique | Peptide Count | Coverage % | Num Unique | Peptide Count | Coverage % | |
BSI0449 Ctrl | 21 | 312 | 12 | 48 | 10.7 | 1 | 2 | 0.8 | 0 | 0 | 0 | 0 | 0 | 0 |
BSI0449 LC | 22 | 509 | 13 | 64 | 13 | 1 | 4 | 0.8 | 0 | 0 | 0 | 0 | 0 | 0 |
BSI0581 Ctrl | 24 | 491 | 3 | 1 | 1.3 | 2 | 7 | 1.7 | 1 | 4 | 1.9 | 0 | 0 | 0 |
BSI0581 LC | 21 | 332 | 3 | 1 | 1.3 | 0 | 0 | 0 | 1 | 4 | 1.9 | 0 | 0 | 0 |
BSI0639 Ctrl | 16 | 139 | 6 | 3 | 4.4 | 1 | 1 | 0.8 | 4 | 14 | 7.7 | 2 | 7 | 3.4 |
BSI0639 LC | 14 | 135 | 0 | 0 | 0 | 0 | 0 | 0 | 5 | 14 | 9.8 | 1 | 2 | 1.9 |
Epitope/mAb | C4A Association | C4A LC vs. Ctrl | HSP90AB1 Association | HSP90AB1 LC vs. Ctrl | 415 gly | 415 gly LC vs. Ctrl | |
---|---|---|---|---|---|---|---|
Proteoform 1 | BSI 449+ | yes | no difference “high” | not detected | not relevant | no difference | LC neutral |
Proteoform 2a | BSI 581+ | yes | no difference “low” | no difference | no difference | different “high in Ctrl” | LC associated |
Proteoform 2b | BSI 581+ | yes | no difference “low” | no difference | no difference | different “low in LC” | LC associated |
Proteoform 3a | BSI 639+ | yes | no difference present in Ctrl only | no difference | no difference | different “high in Ctrl” | LC associated |
Proteoform 3b | BSI 639+ | no | not relevant | no difference | no difference | different “low in LC” | LC associated |
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Tornyi, I.; Lazar, J.; Pettko-Szandtner, A.; Hunyadi-Gulyas, E.; Takacs, L. Epitomics: Analysis of Plasma C9 Epitope Heterogeneity in the Plasma of Lung Cancer Patients and Control Subjects. Int. J. Mol. Sci. 2023, 24, 14359. https://doi.org/10.3390/ijms241814359
Tornyi I, Lazar J, Pettko-Szandtner A, Hunyadi-Gulyas E, Takacs L. Epitomics: Analysis of Plasma C9 Epitope Heterogeneity in the Plasma of Lung Cancer Patients and Control Subjects. International Journal of Molecular Sciences. 2023; 24(18):14359. https://doi.org/10.3390/ijms241814359
Chicago/Turabian StyleTornyi, Ilona, Jozsef Lazar, Aladar Pettko-Szandtner, Eva Hunyadi-Gulyas, and Laszlo Takacs. 2023. "Epitomics: Analysis of Plasma C9 Epitope Heterogeneity in the Plasma of Lung Cancer Patients and Control Subjects" International Journal of Molecular Sciences 24, no. 18: 14359. https://doi.org/10.3390/ijms241814359