Semiautomated high-sensitivity profiling of human blood serum glycoproteins through lectin preconcentration and multidimensional chromatography/tandem mass spectrometry.
We describe an effective analytical approach to identify trace glycoproteins in a small volume of human serum. The system is based on automatable affinity enrichment through silica-based lectin microcolumns and a further separation of the retained glycoproteins on a reversed-phase liquid chromatography with superficially porous packing, operating at high temperature. The fractionated sample is further directed into a 96-well plate for trypsinization and LC-MS/MS analysis. Using a major-component depleted blood serum (16 microg total protein), we were able to identify 271 glycoproteins through this analytical system.
Madera
Milan
M
Department of Chemistry, Indiana University, Bloomington, Indiana 47405, USA.
Mechref
Yehia
Y
Klouckova
Iveta
I
Novotny
Milos V
MV
D002846:Chromatography, Affinity / Q000379:methods; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D037102:Lectins; D013058:Mass Spectrometry / Q000379:methods; D040901:Proteomics / Q000379:methods*; D044967:Serum / Q000737:chemistry*
D006023:Glycoproteins; D037102:Lectins