Enrichment of glycoproteins using nanoscale chelating concanavalin A monolithic capillary chromatography.
Immobilized lectin chromatography can be employed for glycoprotein enrichment, but commonly used columns have limitations of yield and resolution. To improve efficiency and to make the technique applicable to minimal sample material, we have developed a nanoscale chelating Concanavalin A (Con A) monolithic capillary prepared using GMA-EDMA (glycidyl methacrylate-co-ethylene dimethacrylate) as polymeric support. Con A was immobilized on Cu(II)-charged iminodiacetic acid (IDA) regenerable sorbents by forming a IDA:Cu(II):Con A sandwich affinity structure that has high column capacity, as well as stability. When compared with conventional Con A lectin chromatography, the monolithic capillary enabled the better reproducible detection of over double the number of unique N-glycoproteins in human urine samples. Utility for analysis of minimal biological samples was confirmed by the successful elucidation of glycoprotein profiles in mouse urine samples at the microliter scale. The improved efficiency of the nanoscale monolithic capillary will impact the analysis of glycoproteins in complex biological samples, especially where only limited material may be available.
Department of Surgery, The University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
Shun
S
Feng
Na
N
Yang
Subramaniam
S
Pennathur
Steve
S
Goodison
David M
DM
Lubman
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D003208:Concanavalin A / Q000737:chemistry*; D003300:Copper / Q000737:chemistry; D006023:Glycoproteins / Q000302:isolation & purification* / Q000652:urine; D006801:Humans; D007098:Imino Acids / Q000737:chemistry; D051379:Mice
D006023:Glycoproteins; D007098:Imino Acids; D003208:Concanavalin A; D003300:Copper; C008109:iminodiacetic acid