High-sensitivity profiling of glycoproteins from human blood serum through multiple-lectin affinity chromatography and liquid chromatography/tandem mass spectrometry.
We report here the use of high-performance lectin affinity enrichment of glycoproteins at microscale levels using a series of silica-bound lectins. The potential of this approach is being demonstrated for the glycoprotein enrichment from microliter volumes of human blood serum. Individual injections of sample to the affinity microcolumns packed with four lectin materials with different glycan specificities (Con A, SNA-I, UEA-I, PHA-L), followed by off-line reversed-phase pre-fractionation and nano-LC/MS/MS, permitted identification of 108 proteins in the lectin-bound fractions spanning a concentration dynamic range of 7-10 orders of magnitude. In contrast, multi-lectin microcolumn affinity chromatography, an alternative enrichment approach allowed identification of only 67 proteins. An attractive feature of high-performance lectin affinity chromatography at microscale levels is the substantial reduction of sample losses that are commonly experienced with extensive sample preparation needed for larger sample volumes.
Madera
Milan
M
Department of Chemistry, Indiana University, 800 E Kirkwood Ave., Bloomington, IN 47405, United States.
Mechref
Yehia
Y
Klouckova
Iveta
I
Novotny
Milos V
MV
D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D006023:Glycoproteins / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D037102:Lectins; D053719:Tandem Mass Spectrometry / Q000379:methods*
D006023:Glycoproteins; D037102:Lectins