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

Abstract

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 (MS(n)) 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.