Immobilization of aminophenylboronic acid on magnetic beads for the direct determination of glycoproteins by matrix assisted laser desorption ionization mass spectrometry.
Aminophenylboronic acid (APBA) has been immobilized on magnetic beads for the direct determination of glycoprotein by matrix assisted laser desorption/ionizaton time of flight mass spectrometry (MALDI-TOF-MS). An APBA layer was formed on the surface of carboxylic acid terminated magnetic beads by coupling with carbodiimide and subsequently reacted with an N-hydroxysuccinimide moiety. The immobilized APBA was identified by MALDI-TOF-MS without a matrix. Glycoproteins, such as HbA1c, fibrinogen, or RNase B were separated and desalted using APBA magnetic beads by simply washing the magnetic beads and then separating them by external magnet. Proteins can be identified by direct determination of proteins on beads on MALDI plate and confirmed again by peptide mass finger printing after digestion of proteins on magnetic beads by trypsin. Fluorescence image with a FITC tagging protein using confocal laser microscopy showed the difference of immobilization efficiency between glycoproteins and nonglycoproteins. The methods developed within this work allow the simple treatment and enrichment of glycoproteins as well as direct determination of proteins on beads by MALDI-TOF-MS.
Lee
Jeong Heon
JH
Proteonik Research Laboratory, Ansan, South Korea.
Kim
Yangsun
Y
Proteonik Research Laboratory, Ansan, South Korea. yangskim@microbiochip.com.
Ha
Mi Young
MY
Microbiochip Center, Hanyang University, Ansan, South Korea.
Lee
Eun Kyu
EK
Microbiochip Center, Hanyang University, Ansan, South Korea.
Choo
Jaebum
J
Department of Applied Chemistry, Hanyang University, Ansan, South Korea.
D000042:Absorption; D001897:Boronic Acids / Q000737:chemistry*; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry*; D018189:Immunomagnetic Separation / Q000379:methods*; D008863:Microspheres; D011485:Protein Binding; D013048:Specimen Handling / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001897:Boronic Acids; D020099:Coated Materials, Biocompatible; D006023:Glycoproteins; C028592:3-aminobenzeneboronic acid