Characterization of Murine Brain Membrane Glycoproteins by Detergent Assisted Lectin Affinity Chromatography.
Membrane glycoproteins play vital roles in many fundamental physiological and pathophysiological processes in the central nervous system and represent important targets for pharmaceuticals and biomarker discovery. However, their isolation and characterization have been greatly limited. Lectin affinity chromatography (LAC) has evolved as a powerful method to enrich glycoproteins in biofluid and cell/tissue lysate. However, its use in the hydrophobic fraction of the samples has rarely been explored. In this study, we have conducted a systematic investigation on the lectin binding efficiency in the presence of four commonly used detergents. We have found that, under certain concentrations, detergents can minimize nonspecific binding and facilitate the elution of hydrophobic glycoproteins. With the detergent assisted lectin affinity chromatography (DALAC), a total of 1491 proteins were identified with low numbers of false positives from two lectins. Proteins (699) were identified with at least two unique peptides, of which 219 are membrane glycoproteins. Compared to the traditional methods, the DALAC approach significantly increased the recovery of plasma membrane and glycoproteins. NP-40 is recommended as a well rounded detergent for DALAC, but the conditions for enriching certain target proteins need to be empirically determined. This study represents the first global identification of the murine brain glycoproteome.
Wei
Xin
X
Department of Chemistry and School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705.
Dulberger
Charles
C
Li
Lingjun
L