Glycoform composition profiling of O-glycopeptides derived from human serum IgA1 by matrix-assisted laser desorption ionization-time of flight-mass spectrometry.
Pouria, Shideh;
Corran, Patrick H;
Smith, Alice C;
Smith, Howard W;
Hendry, Bruce M;
Challacombe, Stephen J;
Tarelli, Edward;
(2004)
Glycoform composition profiling of O-glycopeptides derived from human serum IgA1 by matrix-assisted laser desorption ionization-time of flight-mass spectrometry.
Analytical biochemistry, 330 (2).
pp. 257-263.
ISSN 0003-2697
DOI: https://doi.org/10.1016/j.ab.2004.03.053
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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.