TransOmic analysis of forebrain sections in Sp2 conditional knockout embryonic mice using IR-MALDESI imaging of lipids and LC-MS/MS label-free proteomics
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Quantitative methods for detection of biological molecules are needed more than ever before in the emerging age of “omics” and “big data.” Here, we provide an integrated approach for systematic analysis of the “lipidome” in tissue. To test our approach in a biological context, we utilized brain tissue selectively deficient for the transcription factor Specificity Protein 2 (Sp2). Conditional deletion of Sp2 in the mouse cerebral cortex results in developmental deficiencies including disruption of lipid metabolism. Silver (Ag) cationization was implemented for infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) to enhance the ion abundances for olefinic lipids, as these have been linked to regulation by Sp2. Combining Ag-doped and conventional IR-MALDESI imaging, this approach was extended to IR-MALDESI imaging of embryonic mouse brains. Further, our imaging technique was combined with bottom-up shotgun proteomic LC-MS/MS analysis and western blot for comparing Sp2 conditional knockout (Sp2-cKO) and wild-type (WT) cortices of tissue sections. This provided an integrated omics dataset which revealed many specific changes to fundamental cellular processes and biosynthetic pathways. In particular, step-specific altered abundances of nucleotides, lipids, and associated proteins were observed in the cerebral cortices of Sp2-cKO embryos.
KeywordsIR-MALDESI Proteomics Lipidomics Mass spectrometry imaging Embryonic brain
This work was supported by NIH R01NS089795 (HTG), NIH R01GM087964 (DCM), and the NIH/NCSU Molecular Biotechnology Training Grant 5T32GM00-8776-08 (PL). FM acknowledges travel funding from DAAD (German Academic Exchange Service).
Compliance with ethical standards
The authors declare that they have no conflicts of interest. Mice used in this study were bred and housed in the College of Veterinary Medicine vivarium according to Institutional Animal Care and Use Committee (IACUC), North Carolina State University regulations, and Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals.
- 16.Loziuk PL, Parker J, Li W, Lin CY, Wang JP, Li Q, et al. Elucidation of xylem-specific transcription factors and absolute quantification of enzymes regulating cellulose biosynthesis in Populus trichocarpa. J Proteome Res. 2015.Google Scholar
- 23.Bray JH, Scott E. Multivariate analysis of variance. Newbury park, CA: Sage; 1985.Google Scholar
- 31.Orth M, Bellosta S. Cholesterol: its regulation and role in central nervous system disorders. Cholesterol. 2012;292598.Google Scholar
- 32.Herz J, Farese Jr RV. The LDL receptor gene family, apolipoprotein B and cholesterol in embryonic development. J Nutr. 1999;2S(Suppl):473S–5S.Google Scholar
- 33.Saher G, Brugger B, Lappe-Siefke C, Mobius W, Tozawa R, Wehr MC, et al. High cholesterol level is essential for myelin membrane growth. Nat Neurosci. 2005;4:468–75.Google Scholar
- 40.Nazari M, Muddiman DC. Polarity switching mass spectrometry imaging of healthy and cancerous hen ovarian tissue sections by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI). Analyst. 2015.Google Scholar
- 41.Laboratories, K. Steroid biosynthesis, KEGG pathway 00100, version 11/29/13. Available: http://www.genome.jp/kegg-bin/show_pathway?map00100
- 45.Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. 1995;7:484–96.Google Scholar
- 57.de Carlos JA, Lopez-Mascaraque L, Valverde F. Dynamics of cell migration from the lateral ganglionic eminence in the rat. J Neurosci. 1996;19:6146–56.Google Scholar
- 62.Tanyi JL, Hasegawa Y, Lapushin R, Morris AJ, Wolf JK, Berchuck A, et al. Role of decreased levels of lipid phosphate phosphatase-1 in accumulation of lysophosphatidic acid in ovarian cancer. Clin Cancer Res. 2003;10(Pt 1):3534–45.Google Scholar
- 64.Jokela H, Rantakari P, Lamminen T, Strauss L, Ola R, Mutka AL, et al. Hydroxysteroid (17beta) dehydrogenase 7 activity is essential for fetal de novo cholesterol synthesis and for neuroectodermal survival and cardiovascular differentiation in early mouse embryos. Endocrinology. 2010;4:1884–92.CrossRefGoogle Scholar
- 66.Gupta S, Knight AG, Keller JN, Bruce-Keller AJ. Saturated long-chain fatty acids activate inflammatory signaling in astrocytes. J Neurochem. 2012;6:1060–71.Google Scholar