Research Article Analysis of mucosal mucins separated by SDS-urea agarose polyacrylamide composite gel electrophoresis Efficient separation of mucins (200 kDa–2 MDa) was demonstrated using gradient SDS agarose/polyacrylamide composite gel electrophoresis (SDS-AgPAGE). Inclusion of urea (SDS-UAgPAGE) in the gels casting were shown to have no effect on the migration of mucins in the gel and allowed casting of gel at room temperature. This simplified the procedure for multiple casting of agarose polyacrylamide gradients and increased reproducibility of these gels. Hence, the implementation of urea makes the technique applicable for high throughput isolation and screening of mucin oligosaccharides by LC-MS after releasing the oligosaccharides from isolated, blotted mucin subpopulations. It was also shown that the urea addition had no effect on other supporting applications such as western and lectin blotting. In addition, identification of the mucin protein after tryptic digestion and LC-MS was possible and no protein carbamylation due to the presence of urea in the gel was detected. LC-MS software developed for metabolomic analysis was used for O-linked oligosaccharide detection and differential display of various mucin samples. Using this method, heterogeneous glycosylation of mucins and mucin-type molecules isolated by SDS-AgPAGE and SDS-UAgPAGE was shown to consist of more than 80 different components in a single band, and in the extreme cases, up to 300–500 components (MUC5B/AC from saliva and sputum and). Metabolomic software was also used to show that the migration of mucin isoforms within the gel is due to heterogeneous size distribution of the oligosaccharides, with the slower migrating bands enriched in high-molecular-weight oligosaccharides. Keywords: Glycomics / Mucin / O-linked glycosylation / Software DOI 10.1002/elps.201100374 1 Introduction Glycosylation is ubiquitous throughout nature and is the major post-translational modification serving many roles in biology, from aiding protein folding and protein stabiliza- tion [1, 2] to structural support [3] and antimicrobial activity [4]. Protein glycosylation is usually found on attached to the nitrogen on asparagines side chain (N-linked glycosylation) or on the oxygen on the side chains of serines and threonines (O-linked glycosylation). Glycosylation of protein has been linked to various disease states and disease progression such as cancer, inflammation bowel diseases and cystic fibrosis [5–8]. Specific carbohydrate structures are involved in tumor metastasis and invasiveness [9], and inflammation/immune functions [10]. O-linked glycosyla- tion is usually clustered into domains rich in Ser/Thr on the protein backbone and are frequently observed on the major proteins on mucosal surfaces, the mucins. Hence, O-linked glycosylation is often referred to as mucin-type glycosyla- tion. Mucin glycosylation on mucosal is a dynamic event changing as a consequence to an ever varying milieu around the mucosal surfaces, a phenomenon also known as legislation [11]. O-linked carbohydrates can account for 80% of the molecular weight of mucins, where the apomucin back- bones are coded for by a family of genes, the MUC (apomucin) genes [12]. Mucins are secreted proteins present on all mucosal surfaces, including the gastrointestinal, reproductive and respiratory tract [12] and are present as large gel-forming mucins and smaller monomeric mucins, where the latter are generally cell membrane-associated [12]. The gel-forming mucins are comprosed of individual mucin subunits joined via disulfide bonds constructing oligomers and polymers [13] making these molecules into macroscopic gel complexes [14]. Treatment with reducing agents releases Samah M. A. Issa 1 Benjamin L. Schulz 2 Nicolle H. Packer 3 Niclas G. Karlsson 4 1 School of Chemistry, National University of Ireland, Galway, Ireland 2 School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia 3 Biomolecular Frontiers Research Centre, Faculty of Science, Macquarie University, Sydney, NSW, Australia 4 Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden Received July 12, 2011 Revised September 23, 2011 Accepted September 23, 2011 Colour Online: See the article online to view Fig. 3 in colour. Abbreviations: IAA, iodoacetamide; PAS, periodic acid-Schiff base staining; WGA, wheat germ agglutinin Correspondence: Dr. Niclas G. Karlsson, University of Gothen- burg, Medical Biochemistry, Box 440, 405 30 Gothenburg, Sweden E-mail: niclas.karlsson@medkem.gu.se Fax: 146-31-416108 & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2011, 32, 3554–3563 3554