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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 3564-3575 |
| Number of pages | 12 |
| Journal | Electrophoresis |
| Volume | 32 |
| Issue number | 24 |
| DOIs | |
| State | Published - Dec 1 2011 |
| Externally published | Yes |
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Keywords
- Biomarker discovery
- Glycoproteome fractionation
- Glycosylation
- Magnetic beads
- Multilectin affinity
ASJC Scopus subject areas
- Analytical Chemistry
- Biochemistry
- Clinical Biochemistry
Cite this
High-throughput lectin magnetic bead array-coupled tandem mass spectrometry for glycoprotein biomarker discovery. / Choi, Eun ju; Loo, Dorothy; Dennis, James W.; O'Leary, Caroline A.; Hill, Michelle M.
In: Electrophoresis, Vol. 32, No. 24, 01.12.2011, p. 3564-3575.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - High-throughput lectin magnetic bead array-coupled tandem mass spectrometry for glycoprotein biomarker discovery
AU - Choi, Eun ju
AU - Loo, Dorothy
AU - Dennis, James W.
AU - O'Leary, Caroline A.
AU - Hill, Michelle M.
PY - 2011/12/1
Y1 - 2011/12/1
N2 - 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.
AB - 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.
KW - Biomarker discovery
KW - Glycoproteome fractionation
KW - Glycosylation
KW - Magnetic beads
KW - Multilectin affinity
UR - http://www.scopus.com/inward/record.url?scp=83755205413&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=83755205413&partnerID=8YFLogxK
U2 - 10.1002/elps.201100341
DO - 10.1002/elps.201100341
M3 - Article
C2 - 22180208
AN - SCOPUS:83755205413
VL - 32
SP - 3564
EP - 3575
JO - Electrophoresis
JF - Electrophoresis
SN - 0173-0835
IS - 24
ER -