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Tandem lectin affinity chromatography monolithic columns with surface immobilised concanavalin A , wheat germ agglutinin and R icinus communis agglutinin‐I for capturing sub‐glycoproteomics from breast cancer and disease‐free human sera
Author(s) -
Selvaraju Subhashini,
El Rassi Ziad
Publication year - 2012
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201200230
Subject(s) - wheat germ agglutinin , concanavalin a , glycoproteomics , lectin , chemistry , glycan , agglutinin , soybean agglutinin , affinity chromatography , glycoprotein , chromatography , biochemistry , enzyme , in vitro
In this study, a liquid‐phase separation platform consisting of tandem lectin affinity chromatography was introduced for the selective capturing of sub‐glycoproteomics that are affected in cancers, e.g. breast cancer. The platform is comprised of three monolithic columns with surface immobilised lectins including concanavalin A ( C on A ), wheat germ agglutinin ( WGA ) and R icinus communis agglutinin‐I ( RCA ‐I). While WGA and C on A have specificities directed towards the core portion of N ‐glycans on the glycoprotein surface, RCA ‐I specifically interacts with the non‐reducing terminal moieties of the outer chain structures of N ‐glycans. The effects of the order in which the three lectin columns were arranged in the tandem columns format were evaluated. The most suitable order proved to be WGA → C on A → RCA ‐I (denoted as WCR ) as far as the number of captured proteins was concerned. The WCR tandem columns allowed the capture of 113 and 112 proteins from disease‐free and breast cancer sera, respectively, corresponding to 75 and 65 non‐redundant proteins, respectively. Using mass spectral count ratios and Q ‐ Q plots yielded a panel of 23 non‐redundant differentially expressed proteins (i.e. a panel of 23 candidate markers), which should in principle be more representative of a pathophysiological state than a single marker candidate.