title: High-throughput lectin magnetic bead array-coupled tandem mass spectrometry for glycoprotein biomarker discovery.
abstract: Alterations in protein glycosylation occur during development and progression of many diseases, hence glycomics and glycoproteomics have emerged as important tools in glycobiomarker discovery. High-throughput glycan profiling can now be achieved with the recent developments in MS-based techniques. To enable identification and rapid monitoring of glycosylation changes in serum proteins, we developed a semi-automated high-throughput glycoprotein biomarker discovery platform termed lectin magnetic bead array-coupled tandem mass spectrometry (LeMBA-MS) which includes (i) effective single-step serum glycoprotein isolation using a panel of 20 individual lectin-coated magnetic beads in microplate format, (ii) on-bead trypsin digestion, and (iii) nanoLC-MS/MS with lectin exclusion list. With use of appropriate sequence databases, LeMBA-MS can detect glycosylation changes regardless of the species. By spiking known amounts of titrated ovalbumin to a serum sample, we report nanomolar sensitivity, and linearity of response of LeMBA-MS using concanavalin A-coupled beads. Neuraminidase treatment led to reduction of binding to sialic acid-binding lectins. Interestingly, we found that desialylation caused increased binding of haptoglobin and hemopexin to mannose-specific lectins, pointing to the importance of identifying a signature of lectin-binding. High-throughput LeMBA-MS to generate glycosylation signatures will facilitate glycobiomarker discovery. LeMBA can be coupled to down-stream detection platforms for validation, making it a truly versatile platform.
authors.affiliation: The University of Queensland Diamantina Institute, Woollongabba, QLD, Australia.
authors.forename: Eunju
authors.initials: E
authors.lastname: Choi
authors.forename: Dorothy
authors.initials: D
authors.lastname: Loo
authors.forename: James W
authors.initials: JW
authors.lastname: Dennis
authors.forename: Caroline A
authors.initials: CA
authors.lastname: O'Leary
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Hill
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000097:blood / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism; D001798:Blood Proteins / Q000737:chemistry; D003208:Concanavalin A / Q000737:chemistry; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D006031:Glycosylation; D057166:High-Throughput Screening Assays; D006801:Humans; D037102:Lectins / Q000737:chemistry / Q000378:metabolism*; D059346:Magnets; D008863:Microspheres; D009439:Neuraminidase / Q000737:chemistry; D040901:Proteomics; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000295:instrumentation* / Q000379:methods
chemical_list: D015415:Biomarkers; D014408:Biomarkers, Tumor; D001798:Blood Proteins; D006023:Glycoproteins; D037102:Lectins; D003208:Concanavalin A; D009439:Neuraminidase