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.

Application of Quality by Design Paradigm to the Manufacture of Protein Therapeutics

“…Previous work has used lectins conjugated to chromatography resin (e.g [175]) or to magnetic beads for recovering glycan containing proteins from a complex mixture, for example serum [176]. The process can be done in small volumes using microtitre plates and the glycoproteins can later be analysed by MS in a process that can be automated by the use of liquid handling apparati [177].…”

Serum diagnostic glycoprotein biomarkers for Barrett's esophagus and esophageal adenocarcinoma

“…Although the study showed promising trends, the statistical power was not achieved due to the very low number of samples. To improve the throughput of glyco-centric proteomics studies, we have developed lectin magnetic bead array-mass spectrometry (LeMBA-MS), a high-throughput platform where a panel of lectins individually immobilized the magnetic beads is used to capture glycoproteins followed by on-bead trypsin digest and liquidchromatography-tandem mass spectrometry for protein identification (317,318). Parallel screening of a panel of lectins may be helpful to identify differentially glycosylated circulating proteins during EAC pathogenesis.…”

“…In order to facilitate high-throughput glycan analysis lectin microarrays have emerged where series of lectins are spotted on the solid support, samples are tagged with fluorophore and detection is carried out using fluorescence microarray scanners (347). Recently our laboratory developed lectin magnetic bead array-tandem mass spectrometry (LeMBA-MS/MS), a high-throughput platform where array of lectins are immobilized on the magnetic beads to capture glycoproteins followed by on bead trypsin digest in line with mass spectrometry (317,318). These lectin based high-throughput techniques have been playing key role in glycobiomarker research.…”

“…Sample preparation for glycoprotein biomarker discovery using LeMBA protocol is semi-automated, reproducible, and high-throughput hence suitable for screening enough number of patient samples required for biomarker discovery (317). Moreover, it uses serum sample as a source of biomarker discovery from beginning of the study (317,318).…”

“…Dynamic exclusion was applied after 2 MS/MS within 0.25 min. Exclusion for lectin peptides was applied as reported previously (317). The QTOF was tuned and calibrated prior to analysis.…”