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.
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.
Wang
Yonghui
Y
Barnett Institute, Northeastern University, Boston, MA 02115, USA.
Wu
Shiaw-Lin
SL
Hancock
William S
WS
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000032:analysis*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D005583:Fourier Analysis; D006023:Glycoproteins / Q000032:analysis* / Q000097:blood; D006031:Glycosylation; D006242:Haptoglobins / Q000032:analysis; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis / Q000097:blood; D037102:Lectins / Q000032:analysis* / Q000097:blood; D008969:Molecular Sequence Data; D009961:Orosomucoid / Q000032:analysis; D010959:Tissue Plasminogen Activator / Q000032:analysis / Q000097:blood
D001798:Blood Proteins; D006023:Glycoproteins; D006242:Haptoglobins; D007074:Immunoglobulin G; D037102:Lectins; D009961:Orosomucoid; D010959:Tissue Plasminogen Activator