Abstract
Aberrant protein glycosylation is a characteristic of diverse diseases which has been explored as biomarkers. To support translational serum glycoprotein biomarker discovery and validation, we developed a semi-automated workflow using individual lectin-coupled magnetic beads to conduct lectin pulldowns in a high-throughput format. Lectins are naturally occurring glycoprotein binding proteins widely used in glycobiology. While lectin-affinity isolation has been coupled to mass spectrometry-based proteomics, the lectin magnetic bead array (LeMBA) platform allows technically robust screening and measurement of clinical cohorts. This chapter describes detailed lectin-magnetic bead coupling, serum denaturation, lectin magnetic bead pulldown, and on-bead trypsin digest. The resulting tryptic peptides are analyzed by untargeted or targeted liquid chromatography-mass spectrometry (LC-MS), for biomarker discovery, or qualification/validation, respectively. LeMBA-MS generates quantitative data for glycoforms based on lectin affinity of the glycoprotein coupled with MS measurement of one or more prototypic peptides and has successfully been used to discover and validate novel serum cancer glycoprotein biomarkers. This chapter includes detailed protocols for two different liquid handlers, along with recommendations on quality control measures for clinical biomarker studies.
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References
Haga Y, Ueda K (2022) Glycosylation in cancer: its application as a biomarker and recent advances of analytical techniques. Glycoconj J 39:303–313
Xu MM et al (2021) Glycoproteins as diagnostic and prognostic biomarkers for neurodegenerative diseases: a glycoproteomic approach. J Neurosci Res 99(5):1308–1324
Yang S, Chatterjee S, Cipollo J (2018) The Glycoproteomics-MS for studying glycosylation in cardiac hypertrophy and heart failure. Proteomics Clin Appl 12(5):e1700075
Liang Y et al (2022) Progress of proteomics driven precision medicine-from a glycosylation view. Rapid Commun Mass Spectrom 36:e9288
Oliveira T et al (2021) The Hitchhiker’s guide to glycoproteomics. Biochem Soc Trans 49(4):1643–1662
Choi E et al (2011) High-throughput lectin magnetic bead array-coupled tandem mass spectrometry for glycoprotein biomarker discovery. Electrophoresis 32(24):3564–3575
Loo D, Jones A, Hill MM (2010) Lectin magnetic bead array for biomarker discovery. J Proteome Res 9(10):5496–5500
Shah AK et al (2015) Serum glycoprotein biomarker discovery and qualification pipeline reveals novel diagnostic biomarker candidates for esophageal adenocarcinoma. Mol Cell Proteomics 14(11):3023–3039
Oungsakul P et al (2021) Candidate glycoprotein biomarkers for canine visceral hemangiosarcoma and validation using semi-quantitative lectin/immunohistochemical assays. Vet Sci 8(3):38
Shah AK et al (2018) Evaluation of serum glycoprotein biomarker candidates for detection of esophageal adenocarcinoma and surveillance of Barrett’s esophagus. Mol Cell Proteomics 17(12):2324–2334
Bringans S et al (2020) A robust multiplex immunoaffinity mass spectrometry assay (PromarkerD) for clinical prediction of diabetic kidney disease. Clin Proteomics 17:37
Whiteaker JR et al (2021) Targeted mass spectrometry enables multiplexed quantification of immunomodulatory proteins in clinical biospecimens. Front Immunol 12:765898
Webster JA et al (2021) Development of EndoScreen Chip, a microfluidic pre-endoscopy triage test for esophageal adenocarcinoma. Cancers (Basel) 13(12):2865
Silva MLS (2019) Lectin biosensors in cancer glycan biomarker detection. Adv Clin Chem 93:1–61
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1 Electronic Supplementary Material
Wash protocol for conjugated magnetic beads performed on the AssayMAP Bravo platform (MP4 297924 kb)
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Dutt, M., Duong, M.N., Bringans, S., Richards, R.S., Lipscombe, R., Hill, M.M. (2023). Semi-Automated Lectin Magnetic Bead Array (LeMBA) for Translational Serum Glycoprotein Biomarker Discovery and Validation. In: Greening, D.W., Simpson, R.J. (eds) Serum/Plasma Proteomics. Methods in Molecular Biology, vol 2628. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2978-9_25
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DOI: https://doi.org/10.1007/978-1-0716-2978-9_25
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-2978-9
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