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Glycobiology Advance Access originally published online on February 23, 2006
Glycobiology 2006 16(6):514-523; doi:10.1093/glycob/cwj091
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© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Approaches to the study of N-linked glycoproteins in human plasma using lectin affinity chromatography and nano-HPLC coupled to electrospray linear ion trap—Fourier transform mass spectrometry

Yonghui Wang1, Shiaw-lin Wu and William S. Hancock2

Barnett Institute, Northeastern University, Boston, MA 02115

1 Present address: Centocor R&D, Inc., 145 King of Prussia Road, Radnor, PA 19087.

2 To whom correspondence should be addressed; e-mail:wi.hancock{at}neu.edu

Received on November 13, 2005; revised on January 23, 2006; accepted on February 13, 2006

In this publication, we will describe the combination of lectin affinity chromatography with nano high performance liquid chromatography (HPLC) coupled to a linear ion trap Fourier transform mass spectrometer (capillary LC-LTQ/FTMS) to characterize N-linked glycosylation structures in human plasma proteins. We used a well-characterized glycoprotein, tissue plasminogen activator (rt-PA), which is present at low levels in blood, as a standard to determine the dynamic range of this approach. N-linked glycopeptides derived from rt-PA could be characterized at a ratio of 1:200 in human plasma (rtPA: total plasma protein, w/w) by accurate mass measurement in the FTMS and fragmentation (MSn) in the linear ion trap. We demonstrated that this platform has the potential to characterize the general N-linked glycosylation structures of abundant glycoproteins present in human plasma without the requirement for antibody-based purification, or additional carbohydrate analytical protocols. This conclusion was supported by the determination of carbohydrate structures for three glycoproteins, IgG, haptoglobin, and alpha-1-acid glycoprotein, at their natural levels in a human plasma sample, but only after the lectin enrichment step.

Key words: Chinese hamster ovary / glycoprotein / mass spectrometry / plasma / lectin


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