ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 330 (2004) 257–263 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2004 Published by Elsevier Inc. doi:10.1016/j.ab.2004.03.053 Glycoform composition proWling of O-glycopeptides derived from human serum IgA1 by matrix-assisted laser desorption ionization-time of Xight-mass spectrometry  Shideh Pouria, a Patrick H. Corran, b Alice C. Smith, c Howard W. Smith, c Bruce M. Hendry, a Stephen J. Challacombe, a and Edward Tarelli d,¤ a GKT School of Medicine and Dentistry, London SE5 9RS, UK b Immunology Unit, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK c Department of Infection, Immunity and InXammation, University of Leicester, Leicester LE5 4PW, UK d Medical Biomics Centre, St. Georges Hospital Medical School, London SW17 ORE, UK Received 9 January 2004 Available online 20 May 2004 Abstract 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 Xight-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 diVerent 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 quantita- tion of the diVerent 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 proWling the O-glycosylation of IgA1. The MS methodology together with the Java data analysis are expected to be generally applicable for proWling O-linked glycopeptides derived from other glycoproteins and probably for N-linked glycopeptide pools.  2004 Published by Elsevier Inc. Keywords: MALDI-ToF-MS; Serum IgA1; O-glycan; Glycopeptide; trypsin; Exoglycosidase Glycosylation is a major posttranslational modiWca- tion of proteins and the roles of glycosylation in health and disease are increasingly important areas of biomedi- cal research. Bacterial and viral infections, for example, are mediated by the interaction of microorganisms with speciWc glycan structures of the host [1] and changes in normal protein glycosylation occurs in rheumatoid arthritis [2], in cancer [3], and in IgA nephropathy [4]. It is the characterization of glycosylation of IgA that is the subject of our investigations. Human serum IgA1 is produced in the bone marrow and the molecule pos- sesses, in addition to two N-glycosylation sites [5,6], an O- glycosylated proline-rich hinge sequence [7,8] (Fig. 1). Alteration in O-glycosylation appears to be a contribut- ing factor to the deposition of immune complexes in the kidney [9] and is the focus of our studies. In normal serum IgA1, O-glycans comprising GalNAc1-, Gal1- 3GalNAc1-, which may be additionally substituted with NeuNAc, fully occupy Thr 228 Ser 230 and Ser 232 and par- tially occupy Thr 225 and Thr 236 (Fig. 1) [8]; in conditions such as IgA nephropathy, a diVerent proWle of glycans may be present [4]. Complete analysis of the O-glycosyla- tion of glycoproteins is not trivial. This is in part due to the availability of just a single highly speciWc O-endo- glycosidase which liberates only the peptide-linked  Financial support from The Charitable Foundation of Guy’s and St Thomas’ Hospitals is gratefully acknowledged. ¤ Corresponding author. Fax: +44-0-20-8725-0245. E-mail address: etarelli@sghms.ac.uk (E. Tarelli).