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Laser capture microdissection–targeted mass spectrometry: a method for multiplexed protein quantification within individual layers of the cerebral cortex


The mammalian neocortex is organized into layers distinguished by the size, packing density, and connectivity of their constituent neurons. Many neuropsychiatric illnesses are complex trait disorders with etiologic factors converging on neuronal protein networks. Cortical pathology of neuropsychiatric diseases, such as schizophrenia, is often restricted to, or more pronounced in, certain cortical layers, suggesting that genetic vulnerabilities manifest with laminar specificity. Thus, the ability to investigate cortical layer-specific protein levels in human postmortem brain is highly desirable. Here, we developed and validated a laser capture microdissection–mass spectrometry (LCM-MS) approach to quantify over 200 proteins in cortical layers 3 and 5 of two cohorts of human subjects as well as a monkey model of postmortem interval. LCM-MS was readily implementable and reliably identified protein patterns that differed between cortical layers 3 and 5. Our findings suggest that LCM-MS facilitates the precise quantification of proteins within individual cortical layers from human postmortem brain tissue, providing a powerful tool in the study of neuropsychiatric disease.

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  1. 1.

    Anderson SA, Eisenstat DD, Shi L, Rubenstein JL. Interneuron migration from basal forebrain to neocortex: dependence on Dlx genes. Science. 1997;278:474–6.

  2. 2.

    Ghashghaei HT, Lai C, Anton ES. Neuronal migration in the adult brain: are we there yet? Nat Rev Neurosci. 2007;8:141–51.

  3. 3.

    Hoftman GD, Datta D, Lewis DA. Layer 3 excitatory and inhibitory circuitry in the prefrontal cortex: developmental trajectories and alterations in schizophrenia. Biol Psychiatry. 2017;81:862–73.

  4. 4.

    Kim EJ, Juavinett AL, Kyubwa EM, Jacobs MW, Callaway EM. Three types of cortical layer 5 neurons that differ in brain-wide connectivity and function. Neuron. 2015;88:1253–67.

  5. 5.

    Tjia M, Yu X, Jammu LS, Lu J, Zuo Y. Pyramidal neurons in different cortical layers exhibit distinct dynamics and plasticity of apical dendritic spines. Front Neural Circuits. 2017;11:43.

  6. 6.

    Sullivan PF, Daly MJ, O’Donovan M. Genetic architectures of psychiatric disorders: the emerging picture and its implications. Nat Rev Genet. 2012;13:537–51.

  7. 7.

    Harrison PJ, West VA. Six degrees of separation: on the prior probability that schizophrenia susceptibility genes converge on synapses, glutamate and NMDA receptors. Mol Psychiatry. 2006;11:981–3.

  8. 8.

    Banerjee A, Macdonald ML, Borgmann-Winter KE, Hahn CG. Neuregulin 1-erbB4 pathway in schizophrenia: from genes to an interactome. Brain Res Bull. 2010;83:132–9.

  9. 9.

    Moyer CE, Shelton MA, Sweet RA. Dendritic spine alterations in schizophrenia. Neurosci Lett. 2015;601:46–53.

  10. 10.

    Sweet RA, Fish KN, Lewis DA. Mapping synaptic pathology within cerebral cortical circuits in subjects with schizophrenia 2. Front Hum Neurosci. 2010;4:44.

  11. 11.

    Arion D, Horvath S, Lewis DA, Mirnics K. Infragranular gene expression disturbances in the prefrontal cortex in schizophrenia: signature of altered neural development? Neurobiol Dis. 2010;37:738–46.

  12. 12.

    Arion D, Unger T, Lewis DA, Mirnics K. Molecular markers distinguishing supragranular and infragranular layers in the human prefrontal cortex. Eur J Neurosci. 2007;25:1843–54.

  13. 13.

    Arion D, Corradi JP, Tang S, Datta D, Boothe F, He A, et al. Distinctive transcriptome alterations of prefrontal pyramidal neurons in schizophrenia and schizoaffective disorder. Mol Psychiatry. 2015;20:1397–405.

  14. 14.

    Altar CA, Jurata LW, Charles V, Lemire A, Liu P, Bukhman Y, et al. Deficient hippocampal neuron expression of proteasome, ubiquitin, and mitochondrial genes in multiple schizophrenia cohorts. Biol Psychiatry. 2005;58:85–96.

  15. 15.

    de Sousa Abreu R, Penalva LO, Marcotte EM, Vogel C. Global signatures of protein and mRNA expression levels. Mol Biosyst. 2009;5:1512–26.

  16. 16.

    Zhang B, Gaiteri C, Bodea LG, Wang Z, McElwee J, Podtelezhnikov AA, et al. Integrated systems approach identifies genetic nodes and networks in late-onset Alzheimer’s disease. Cell. 2013;153:707–20.

  17. 17.

    Sharma K, Schmitt S, Bergner CG, Tyanova S, Kannaiyan N, Manrique-Hoyos N, et al. Cell type- and brain region-resolved mouse brain proteome. Nat Neurosci. 2015;18:1819–31.

  18. 18.

    Mukherjee S, Rodriguez-Canales J, Hanson J, Emmert-Buck MR, Tangrea MA, Prieto DA, et al. Proteomic analysis of frozen tissue samples using laser capture microdissection. Methods Mol Biol. 2013;1002:71–83.

  19. 19.

    Heath JR, Ribas A, Mischel PS. Single-cell analysis tools for drug discovery and development. Nat Rev Drug Discov. 2016;15:204–16.

  20. 20.

    Nijholt DA, Stingl C, Luider TM. Laser capture microdissection of fluorescently labeled amyloid plaques from Alzheimer’s disease brain tissue for mass spectrometric analysis. Methods Mol Biol. 2015;1243:165–73.

  21. 21.

    Hashimoto M, Bogdanovic N, Nakagawa H, Volkmann I, Aoki M, Winblad B, et al. Analysis of microdissected neurons by 18O mass spectrometry reveals altered protein expression in Alzheimer’s disease. J Cell Mol Med. 2012;16:1686–700.

  22. 22.

    MacDonald ML, Ciccimaro E, Prakash A, Banerjee A, Seeholzer SH, Blair IA, et al. Biochemical fractionation and stable isotope dilution liquid chromatography-mass spectrometry for targeted and microdomain-specific protein quantification in human postmortem brain tissue. Mol Cell Proteom. 2012;11:1670–81.

  23. 23.

    Andressen C, Blumcke I, Celio MR. Calcium-binding proteins: selective markers of nerve cells. Cell Tissue Res. 1993;271:181–208.

  24. 24.

    Glantz LA, Lewis DA. Decreased dendritic spine density on prefrontal cortical pyramidal neurons in schizophrenia. Arch General Psychiatry. 2000;57:65–73.

  25. 25.

    Curley AA, Arion D, Volk DW, Asafu-Adjei JK, Sampson AR, Fish KN, et al. Cortical deficits of glutamic acid decarboxylase 67 expression in schizophrenia: clinical, protein, and cell type-specific features. Am J Psychiatry. 2011;168:921–9.

  26. 26.

    Hayes TL, Cameron JL, Fernstrom JD, Lewis DA. A comparative analysis of the distribution of prosomatostatin-derived peptides in human and monkey neocortex. J Comp Neurol. 1991;303:584–99.

  27. 27.

    Datta D, Arion D, Roman KM, Volk DW, Lewis DA. Altered expression of ARP2/3 complex signaling pathway genes in prefrontal layer 3 pyramidal cells in schizophrenia. Am J Psychiatry. 2017;174:163–71.

  28. 28.

    Datta D, Arion D, Corradi JP, Lewis DA. Altered expression of CDC42 signaling pathway components in cortical layer 3 pyramidal cells in schizophrenia. Biol Psychiatry. 2015;78:775–85.

  29. 29.

    Bayes A, van de Lagemaat LN, Collins MO, Croning MD, Whittle IR, Choudhary JS, et al. Characterization of the proteome, diseases and evolution of the human postsynaptic density. Nat Neurosci. 2011;14:19–21.

  30. 30.

    MacDonald ML, Ding Y, Newman J, Hemby S, Penzes P, Lewis DA, et al. Altered glutamate protein co-expression network topology linked to spine loss in the auditory cortex of schizophrenia. Biol Psychiatry. 2015;77:959–68.

  31. 31.

    MacLean B, Tomazela DM, Shulman N, Chambers M, Finney GL, Frewen B, et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 2010;26:966–8.

  32. 32.

    Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA. 1998;95:14863–8.

  33. 33.

    Page RD. TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci CABIOS. 1996;12:357–8.

  34. 34.

    Hayes TL, Lewis DA. Nonphosphorylated neurofilament protein and calbindin immunoreactivity in layer III pyramidal neurons of human neocortex. Cereb Cortex. 1992;2:56–67.

  35. 35.

    Method of the Year 2012, Nat Methods. 2013;10:1.

  36. 36.

    Uhlen M, Fagerberg L, Hallstrom BM, Lindskog C, Oksvold P, Mardinoglu A, et al. Proteomics. Tissue-based map of the human proteome. Science. 2015;347:1260419.

  37. 37.

    Ping L, Duong DM, Yin L, Gearing M, Lah JJ, Levey AI, et al. Global quantitative analysis of the human brain proteome in Alzheimer’s and Parkinson’s Disease. Sci Data. 2018;5:180036.

  38. 38.

    Kim DK, Park J, Han D, Yang J, Kim A, Woo J, et al. Molecular and functional signatures in a novel Alzheimer’s disease mouse model assessed by quantitative proteomics. Mol Neurodegener. 2018;13:2.

  39. 39.

    Angelo M, Bendall SC, Finck R, Hale MB, Hitzman C, Borowsky AD, et al. Multiplexed ion beam imaging of human breast tumors. Nat Med. 2014;20:436–42.

  40. 40.

    Ornatsky O, Baranov VI, Bandura DR, Tanner SD, Dick J. Multiple cellular antigen detection by ICP-MS. J Immunol Methods. 2006;308:68–76.

  41. 41.

    Mizee MR, Miedema SS, van der Poel M, Adelia, Schuurman KG, van Strien ME, et al. Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables. Acta Neuropathol Commun. 2017;5:16.

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Correspondence to Matthew L. MacDonald.

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