Modification of aluminum chips for LDI mass spectrometry of proteins.
Matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI TOFMS) combined with affinity chromatography on immobilized phenylboronic acid agarose gels was used for selective enrichment and detection of specifically modified proteins such as glycated proteins in complex biological samples. Physicochemical grafting of hydrophilic polymers on aluminum surface was developed to reduce nonspecific protein sorption and to create a proper support layer for a three-dimensional affinity hydrogel. Grafted agarose allowed the fixation of three-dimensional agarose hydrogel on the chip surface. Both pinched polymers and hydrogels were effectively derivatized. 3-Aminophenylboronic acid (mPBA) was covalently immobilized as an affinity ligand to achieve specific binding of glycated plasma proteins. Alternatively, the affinity sorbent was immersed into the hydrogel to increase binding capacity. MALDI TOFMS was used to evaluate binding efficiency and molecular mass changes of human serum albumin due to glycation. Glycated proteins were captured directly on the chip with high selectivity and efficacy, and low nonspecific binding. Thus they could easily be characterized by MALDI TOFMS.
Shmanai
Vadim
V
BioAnalyt GmbH, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany. shmanai@ifoch.bas-net.by
Gontarev
Sergey
S
Frey
Simone K
SK
Schweigert
Florian J
FJ
D000535:Aluminum / Q000737:chemistry*; D001897:Boronic Acids / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D004983:Ethanolamines / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D017127:Glycation End Products, Advanced; D006801:Humans; D020100:Hydrogels / Q000737:chemistry; D011142:Polyvinyl Alcohol / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D012709:Serum Albumin / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D000093423:Glycated Serum Albumin
D001897:Boronic Acids; D004983:Ethanolamines; D005452:Fluoresceins; D017127:Glycation End Products, Advanced; D020100:Hydrogels; D011506:Proteins; D012709:Serum Albumin; C028592:3-aminobenzeneboronic acid; C024098:6-carboxyfluorescein; C035597:phenylboronic acid-sepharose; D011142:Polyvinyl Alcohol; D012685:Sepharose; C009546:triethanolamine; D000535:Aluminum; D000093423:Glycated Serum Albumin