Developmentally-Dynamic Murine Brain Proteomes and Phosphoproteomes Revealed by Quantitative Proteomics
Abstract
:1. Introduction
2. Experimental
2.1. Tissue Harvesting and Preparation of Tryptic Peptides
2.2. Dimethyl Labeling of Tryptic Peptides
2.3. Anti-Phosphotyrosine Peptide Immunoprecipitations
2.4. SCX-IMAC
2.5. Mass Spectrometry and Data Analysis
2.6. Immunoblots and Antibodies
2.7. Bioinformatics
3. Results and Discussion
3.1. Comparative Murine Brain Proteomics Reveals Developmentally-Dynamic Proteomes and Phosphoproteomes
3.2. Immunoblotting of Developmentally-Important Phosphoproteins Shows Agreement with Quantitative MS Data
3.3. Proteomes and Phosphoproteomes Display Distinct Profiles at Different Stages of Brain Development
3.4. Phosphotyrosine Sites Showing the Most Extreme Differences between E16.5 and P21 Brain Offer Avenues for Distinct Hypothesis Testing
Gene Symbol | pY Site | Peptide (Y# is pY) | Log2 Fold Higher E16.5 over P0 | Fold Higher E16.5 over P0 | Protein Name |
Mbp | 199 | TTHY#GSLPQK | 6.61 | 98 | Myelin basic protein |
Dlg4 | 283 | NTYDVVY#LK | 6.35 | 81 | Discs large homolog 4, Postsynaptic density protein 95 |
Syt1 | 229 | TLVMAVY#DFDR | 5.51 | 45 | Synaptotagmin I |
Grin2b | 1039 | HSQLSDLY#GK | 5.39 | 42 | GluRepsilon2/ N-methyl D-aspartate receptor 2B |
Dlg2 | 340 | HMLGEDDY#TRPPEPVYSTVNK | 4.92 | 30 | Discs large homolog 2, Postsynaptic density protein 93 |
Syt1 | 380 | VFVGY#NSTGAELR | 4.54 | 23 | Synaptotagmin I |
Ckmt1 | 154 | SGY#FDER | 4.53 | 23 | Creatine kinase, mitochondrial 1 |
Mbp | 169 | GAY#DAQGTLSK | 3.64 | 12 | Myelin basic protein |
Dlg4 | 647 | FIEAGQY#NSHLYGTSVQSVR | 3.50 | 11 | Discs large homolog 4, Postsynaptic density protein 95 |
Gene Symbol | pY Site | Peptide (Y# is pY) | Log2 Fold Higher P0 over P21 | Fold Higher P0 over P21 | Protein Name |
Dab1 | 232 | EGVY#DVPK | 3.91 | 15 | Disabled 1 |
Prpf4b | 338 | LCDFGSASHVADNDITPY#LVSR | 3.99 | 16 | Pre-mRNA-processing factor 4b |
Afap1l2 | 413 | VAQQPLSLVGCDVLPDPSPDHLY#SFR | 4.21 | 18 | Actin filament-associated protein 1-like 2 |
Hipk3 | 359 | TVCSTY#LQSR | 4.58 | 24 | Homeodomain interacting protein kinase 3 |
Gsk3b | 216 | GEPNVSY#ICSR | 4.64 | 25 | Glycogen synthase kinase-3 beta |
Hipk1 | 352 | AVCSTY#LQSR | 5.23 | 38 | Homeodomain interacting protein kinase 1 |
3.5. Bioinformatics Reveals Functional and Developmental Dynamics in Brain Proteomes and Phosphoproteomes
Enriched Gene Ontology Categories of Phosphoproteins Showing Major Fold Changes in the E16.5:P0 Phosphopeptide Comparison | |
Enriched in E16.5 | Enriched in P0 |
Cytoskeleton | Protein Complexes Cytoskeleton Non-Membrane Bound Organelles |
Neuronal Projection | |
Synaptic Transmission | |
Cell Signaling | |
Splicing | |
Enriched Gene Ontology Categories of Phosphoproteins Showing Major Fold Changes in the P0:P21 Phosphopeptide Comparison | |
Enriched in P0 | Enrichedin P21 |
Neurogenesis and Differentiation | Synaptic Transmission |
Developmental Growth | Ion Transport |
Morphogenesis | Cognition |
Cell Signaling | Cell Signaling |
Various Metabolism | Non-Membrane Bound Organelles |
4. Conclusions
Supplementary Materials
Supplementary File 1Acknowledgments
Author Contributions
Conflicts of Interest
References
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Doubleday, P.F.; Ballif, B.A. Developmentally-Dynamic Murine Brain Proteomes and Phosphoproteomes Revealed by Quantitative Proteomics. Proteomes 2014, 2, 191-207. https://doi.org/10.3390/proteomes2020191
Doubleday PF, Ballif BA. Developmentally-Dynamic Murine Brain Proteomes and Phosphoproteomes Revealed by Quantitative Proteomics. Proteomes. 2014; 2(2):191-207. https://doi.org/10.3390/proteomes2020191
Chicago/Turabian StyleDoubleday, Peter F., and Bryan A. Ballif. 2014. "Developmentally-Dynamic Murine Brain Proteomes and Phosphoproteomes Revealed by Quantitative Proteomics" Proteomes 2, no. 2: 191-207. https://doi.org/10.3390/proteomes2020191