Deepening into Intracellular Signaling Landscape through Integrative Spatial Proteomics and Transcriptomics in a Lymphoma Model
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Cultures
2.2. Protein Extraction and Quantification
2.2.1. Protocol #1
2.2.2. Protocol #2
2.2.3. Protocol #3
2.2.4. Protocol #4
2.2.5. Protocol #5
2.2.6. Protocol #6
2.2.7. Protocol #7
2.2.8. Protocol #8
2.2.9. Protocol #9
2.3. Proteomics Analysis
2.3.1. Protein Digestion and LC-MS/MS Analysis
2.3.2. Database Search
2.3.3. Quantitative Analysis of MS/MS Datasets
2.3.4. RNA-Sequencing Transcriptomics
2.4. Biotin Protein Labeling
2.5. Size Exclusion Chromatography (SEC): Fast Protein Liquid Chromatography (FPLC)
2.6. Protein Microarrays
2.7. Evaluation of Array Performance at Different MW Fractions
2.8. Image Analysis and Data Acquisition
2.9. Protein Microarray Data Processing
Normalization
2.10. SEC-MAP Database
2.11. Integration of Transcriptomics, Proteomics, and SEC-MAP Datasets
2.12. Visualization of Transcriptomics, Proteomics, and SEC-MAP Datasets
3. Results
3.1. Protein Extraction Strategies for Multi-Pronged Proteomics Characterization
Performance of SEC-MAP: Effect of Protein Extraction Procedures and Biotin Conjugation
3.2. Deciphering Differential Protein Profiles by SEC-MAP
3.2.1. Analysis of Intracellular Signaling Pathways by SEC-MAP
3.2.2. Multi-Protein Complex Analysis by SEC-MAP
3.2.3. Analysis of Specific Protein Isoforms and/or Variants by SEC-MAP
3.2.4. Orthogonal Integration of SEC-MAP with Multi-Omics Datasets (RNA-Seq & LC-MS/MS)
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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Protocols | ||||||||||||||
Buffer elements | #1 | #2 | #3 | #4 | #5 | #6 | #7 | #8 | #9 | |||||
Step 1 | Phosphatase Inhibitor (mM) | TCEP | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | ||
PMSF | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | ||||
NaF | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | ||||
Sodium orthovanadate | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | ||||
β-glycerophosphate | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | ||||
Sodium pyrophosphate tetrabasic decahydrate | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | ||||
Salt (mM) | NaCl | 140 | - | - | 140 | - | - | 140 | - | - | 400 | |||
KCl | - | - | - | - | 15 | 15 | - | 15 | 15 | - | ||||
Nuclear envelope protector (mM) | MgCl2 | - | - | - | 10 | 2 | 2 | 10 | 2 | 2 | 2 | |||
Metalloproteinase inhibitor (mM) | EDTA | 50 | - | - | - | 1 | 1 | - | 1 | 1 | 1 | |||
Chaotropic agent (M) | Urea | - | 9 | 7 | - | - | - | - | - | - | - | |||
Thiourea | - | - | 2 | - | - | - | - | - | - | - | ||||
Buffer solution (mM) | Tris/HCl | 20 | - | 30 | - | - | - | - | - | - | - | |||
HEPES | 20 | - | 5 | 30 | 30 | 5 | 30 | 30 | 30 | |||||
Thickening agent (%-v/v-) | Glycerol | 10 | - | - | - | 20 | 20 | - | 20 | 20 | - | |||
Non ionic detergent (%-v/v-) | IGEPAL | 1 | - | - | - | - | - | - | - | - | - | |||
Tween 20 | - | - | - | 0.1 | - | - | 0.1 | - | - | - | ||||
Laurylmaltoside | - | - | - | - | 10 | - | - | - | - | - | ||||
Triton X-100 | - | - | - | - | - | 1.5 | - | - | - | - | ||||
Protocols | ||||||||||||||
Buffer elements | #1 | #2 | #3 | #4 | #5 | #6 | #7 | #8 | #9 | |||||
Step 1 | Non ionic detergent (%-v/v-) | Digitonin | - | - | - | - | - | - | - | 0.015 | 0.015 | - | ||
Octyl-β-D-glucopiranoside | - | - | - | - | - | - | 38 | - | - | - | ||||
Step 2 | Non ionic detergent (%-v/v-) | Tween 20 | - | - | - | - | - | - | - | 5 | 5 | - | ||
Step 3 | Salt (mM) | NaCl | - | - | - | - | - | - | - | 14 | - | - | ||
Non ionic detergent (%-v/v-) | IGEPAL | - | - | - | - | - | - | - | - | - | 0.5 | |||
Step 4 | Laurylmaltoside | - | - | - | - | - | - | - | 1 | - | 1 | |||
Centrifugation | Time (min) | 15 | 15 | 15 | 15 | 5 | 5 | 15 | 5 | 5 | 5 | |||
103× g | 15 | 15 | 12 | 12 | 16 | 16 | 15 | 0.5 | 0.5 | 3/15 | ||||
Legend | ||||||||||||||
Phosphatase Inhibitor | Salt | Nuclear envelope protector | Metalloproteinase inhibitor | |||||||||||
Chaotropic agent | Buffer solution | Thickening agent | Non ionic detergent | |||||||||||
Centrifugation |
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Landeira-Viñuela, A.; Díez, P.; Juanes-Velasco, P.; Lécrevisse, Q.; Orfao, A.; De Las Rivas, J.; Fuentes, M. Deepening into Intracellular Signaling Landscape through Integrative Spatial Proteomics and Transcriptomics in a Lymphoma Model. Biomolecules 2021, 11, 1776. https://doi.org/10.3390/biom11121776
Landeira-Viñuela A, Díez P, Juanes-Velasco P, Lécrevisse Q, Orfao A, De Las Rivas J, Fuentes M. Deepening into Intracellular Signaling Landscape through Integrative Spatial Proteomics and Transcriptomics in a Lymphoma Model. Biomolecules. 2021; 11(12):1776. https://doi.org/10.3390/biom11121776
Chicago/Turabian StyleLandeira-Viñuela, Alicia, Paula Díez, Pablo Juanes-Velasco, Quentin Lécrevisse, Alberto Orfao, Javier De Las Rivas, and Manuel Fuentes. 2021. "Deepening into Intracellular Signaling Landscape through Integrative Spatial Proteomics and Transcriptomics in a Lymphoma Model" Biomolecules 11, no. 12: 1776. https://doi.org/10.3390/biom11121776
APA StyleLandeira-Viñuela, A., Díez, P., Juanes-Velasco, P., Lécrevisse, Q., Orfao, A., De Las Rivas, J., & Fuentes, M. (2021). Deepening into Intracellular Signaling Landscape through Integrative Spatial Proteomics and Transcriptomics in a Lymphoma Model. Biomolecules, 11(12), 1776. https://doi.org/10.3390/biom11121776