Lectin magnetic bead array for biomarker discovery.
Alterations in protein glycosylation play an important role in patho-physiology, and much effort has been devoted to detecting glycoprotein biomarkers. In this manuscript, we describe the development of a novel method for monitoring alterations in protein glycosylation. Lectins are used as individual affinity reagents and coupled to magnetic beads (Dynabeads) in a microplate array format for isolation of glycosylated proteins. Isolated glycoproteins are digested with trypsin in-solution followed by LC-MS/MS, allowing a liquid handler-assisted high throughput workflow. We demonstrate the specific and reproducible affinity-isolation of glycoproteins using the lectin Dynabead array technology. When used with serum, we achieved one-step purification of glycoproteins with minimal coisolation of abundant serum proteins including albumin. We further optimized the proteomics workflow to allow transfer to a liquid handler for automation. In summary, we report the development of a high throughput platform to detect alterations in protein glycosylation which will be useful in glycoproteomics studies, particularly clinical proteomics studies where large sample sizes are required to achieve statistical power.
Loo
Dorothy
D
The University of Queensland Diamantina Institute, Princess Alexandra Hospital, Brisbane, Australia.
Jones
Alun
A
Hill
Michelle M
MM
D015415:Biomarkers / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000378:metabolism*; D008280:Magnetics; D013058:Mass Spectrometry; D008863:Microspheres; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
D015415:Biomarkers; D006023:Glycoproteins; D037102:Lectins