Tandem lectin affinity chromatography monolithic columns with surface immobilised concanavalin A, wheat germ agglutinin and Ricinus communis agglutinin-I for capturing sub-glycoproteomics from breast cancer and disease-free human sera.
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 (Con A), wheat germ agglutinin (WGA) and Ricinus communis agglutinin-I (RCA-I). While WGA and Con 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 → Con 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.
Department of Chemistry, Oklahoma State University, Stillwater, OK 74078-3071, USA.
Subhashini
S
Selvaraju
Ziad
Z
El Rassi
D001943:Breast Neoplasms / Q000097:blood / Q000737:chemistry* / Q000175:diagnosis; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D003208:Concanavalin A / Q000737:chemistry*; D005260:Female; D006023:Glycoproteins / Q000097:blood / Q000302:isolation & purification*; D006801:Humans; D007700:Kinetics; D037102:Lectins / Q000737:chemistry*; D037121:Plant Lectins / Q000737:chemistry*; D014909:Wheat Germ Agglutinins / Q000737:chemistry*
D006023:Glycoproteins; D037102:Lectins; D037121:Plant Lectins; C017707:Ricinus communis agglutinin-1; D014909:Wheat Germ Agglutinins; D003208:Concanavalin A