Glycoform composition profiling of O-glycopeptides derived from human serum IgA1 by matrix-assisted laser desorption ionization-time of flight-mass spectrometry.
Pools of O-glycopeptides prepared from trypsin-digested reduced and alkylated human serum IgA1 have been analyzed using matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-ToF-MS) in the positive-ion mode, using 2,4,6-trihydroxy acetophenone-ammonium citrate matrix. Dozens of such pools prepared from normal serum IgA1 and from serum of patients with a number of different medical conditions have been routinely analyzed in this manner. The glycopeptides present in these pools possess identical amino acid sequences but are substituted with a variety of neutral and sialylated glycans and the spectra obtained were such that individual compositional glycoforms were baseline resolved. In addition, the spectra were reproducible, exhibiting a relative peak intensity and area variation of around 11-16%, enabling the technique to be used for the relative quantitation of the different compositional glycoforms present. This could be achieved manually or by applying a Java program especially developed for this purpose. The MS analysis described here is a major improvement over present MALDI methods used for profiling the O-glycosylation of IgA1. The MS methodology together with the Java data analysis are expected to be generally applicable for profiling O-linked glycopeptides derived from other glycoproteins and probably for N-linked glycopeptide pools.
Pouria
Shideh
S
GKT School of Medicine and Dentistry, London SE5 9RS, UK.
Corran
Patrick H
PH
Smith
Alice C
AC
Smith
Howard W
HW
Hendry
Bruce M
BM
Challacombe
Stephen J
SJ
Tarelli
Edward
E
D006020:Glycopeptides / Q000032:analysis*; D006801:Humans; D007070:Immunoglobulin A / Q000097:blood / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D006020:Glycopeptides; D007070:Immunoglobulin A; D011134:Polysaccharides