Abstract
Glycosylation is the most common posttranslational modification of proteins in mammalian cells and is limited mainly to membrane and secreted proteins. Glycoproteins play several key roles in the physiology and pathophysiology of the blood-brain barrier (BBB) and are attractive as diagnostic markers and therapeutic targets for many neurological diseases. However, large-scale glycoproteomic studies of the BBB have been lacking, largely due to the complexity of analyzing glycoproteins and a lack of available tools for this analysis. Recent development of the hydrazide capture method and significant advances in mass spectrometry (MS)-based proteomics over the last few years have enabled selective enrichment of glycoproteins from complex biological samples and their quantitative comparisons in multiple conditions. In this chapter, we describe methods for: (1) isolating membrane and secreted proteins from BEC and other cells of the neurovascular unit, (2) enriching glycoproteins using hydrazide capture, and (3) performing label-free quantitative proteomics to identify differential glycoprotein expression in various biological conditions. Hydrazide capture, when coupled with label-free quantitative proteomics, is a reproducible and sensitive method that allows for quantitative profiling of a large number of glycoproteins from biological samples for the purposes of differential expression measurements and biomarker discovery.
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References
Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME (2008) Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, Plainview
Begley DJ, MW Brightman (2003) Structural and functional aspects of the blood-brain barrier. Prog Drug Res 61, 39–78
Lawrenson JG, Cassella JP, Hayes AJ, Firth JA, Allt G (2000) Endothelial glycoconjugates: a comparative lectin study of the brain, retina and myocardium. J Anat 196 (Pt 1), 55–60
Kumagai AK, Dwyer KJ, Pardridge WM (1994) Differential glycosylation of the GLUT1 glucose transporter in brain capillaries and choroid plexus. Biochim Biophys Acta 1193, 24–30
Michel CC (1996) Transport of macromolecules through microvascular walls. Cardiovasc Res 32, 644–653
Vogel J, Sperandio M, Pries AR, Linderkamp O, Gaehtgens P, Kuschinsky W (2000) Influence of the endothelial glycocalyx on cerebral blood flow in mice. J Cereb Blood Flow Metab 20, 1571–1578
Weinbaum S, Tarbell JM, Damiano ER (2007) The structure and function of the endothelial glycocalyx layer. Annu Rev Biomed Eng 9, 121–167
Weinbaum S, Zhang X, Han Y, Vink H, Cowin SC (2003) Mechanotransduction and flow across the endothelial glycocalyx. Proc Natl Acad Sci U S A 100, 7988–7995
Krum JM, More NS, Rosenstein JM (1991) Brain angiogenesis: variations in vascular basement membrane glycoprotein immunoreactivity. Exp Neurol 111, 152–165
Man S, Ubogu EE, Ransohoff RM (2007) Inflammatory cell migration into the central nervous system: a few new twists on an old tale. Brain Pathol 17, 243–250
Cayrol R, Wosik K, Berard JL, Dodelet-Devillers A, Ifergan I, Kebir H, Haqqani AS, Kreymborg K, Krug S, Moumdjian R, Bouthillier A, Becher B, Arbour N, David S, Stanimirovic D, Prat A (2008) Activated leukocyte cell adhesion molecule promotes leukocyte trafficking into the central nervous system. Nat Immunol 9, 137–145
Zamze S, Harvey DJ, Pesheva P, Mattu TS, Schachner M, Dwek RA, Wing DR (1999) Glycosylation of a CNS-specific extracellular matrix glycoprotein, tenascin-R, is dominated by O-linked sialylated glycans and “brain-type” neutral N-glycans. Glycobiology 9, 823–831
Hughes RC (1992) Role of glycosylation in cell interactions with extracellular matrix. Biochem Soc Trans 20, 279–284
Vorbrodt AW, Dobrogowska DH, Lossinsky AS, Wisniewski HM (1986) Ultrastructural localization of lectin receptors on the luminal and abluminal aspects of brain micro-blood vessels. J Histochem Cytochem 34, 251–261
Zhang H, Li XJ, Martin DB, Aebersold R (2003) Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol 21, 660–666
Sun B, Ranish JA, Utleg AG, White JT, Yan X, Lin B, Hood L (2007) Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics. Mol Cell Proteomics 6, 141–149
Weksler BB, Subileau EA, Perriere N, Charneau P, Holloway K, Leveque M, Tricoire-Leignel H, Nicotra A, Bourdoulous S, Turowski P, Male DK, Roux F, Greenwood J, Romero IA, Couraud PO (2005) Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19, 1872–1874
Hill JJ, Moreno MJ, Lam JC, Haqqani AS, Kelly JF (2009) Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label-free quantification. Proteomics 9, 535–549
Palagi PM, Walther D, Quadroni M, Catherinet S, Burgess J, Zimmermann-Ivol CG, Sanchez JC, Binz PA, Hochstrasser DF, Appel RD (2005) MSight: an image analysis software for liquid chromatography-mass spectrometry. Proteomics 5, 2381–2384
Hirosawa M, Hoshida M, Ishikawa M, Toya T (1993) MASCOT: multiple alignment system for protein sequences based on three-way dynamic programming. Comput Appl Biosci 9, 161–167
Haqqani AS, Kelly JF, Stanimirovic DB (2008) Quantitative protein profiling by mass spectrometry using label-free proteomics. Methods Mol Biol 439, 241–256
Haqqani AS, Hutchison JS, Ward R, Stanimirovic DB (2007) Biomarkers and diagnosis; protein biomarkers in serum of pediatric patients with severe traumatic brain injury identified by ICAT-LC-MS/MS. J Neurotrauma 24, 54–74
Acknowledgments
Thanks are expressed to Dr. P.O. Couraud and his group (Institut Cochin, Université Paris Descartes, Paris, France) for providing the immortalized HBEC cells (hCMEC/D3) used in this study.
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Haqqani, A.S., Hill, J.J., Mullen, J., Stanimirovic, D.B. (2011). Methods to Study Glycoproteins at the Blood-Brain Barrier Using Mass Spectrometry. In: Nag, S. (eds) The Blood-Brain and Other Neural Barriers. Methods in Molecular Biology, vol 686. Humana Press. https://doi.org/10.1007/978-1-60761-938-3_16
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DOI: https://doi.org/10.1007/978-1-60761-938-3_16
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