Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ ionization-time of flight-mass spectrometry.
Peptide mass mapping analysis, utilizing a regenerable enzyme microreactor with metal-ion chelated adsorption of enzyme, combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was developed. Different procedures from the conventional approaches were adopted to immobilize the chelator onto the silica supports, that is, the metal chelating agent of iminodiacetic acid (IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the inner wall of the fused-silica capillary pretreated with NH(4)HF(2). The metal ion of copper and subsequently enzyme was specifically adsorbed onto the surface to form the immobilized enzyme capillary microreactor, which was combined with MALDI-TOF-MS to apply for the mass mapping analysis of nL amounts of protein samples. The results revealed that the peptide mapping could routinely be generated from 0.5 pmol protein sample in 15 min at 50 degrees C, even 20 fmol cytochrome c could be well digested and detected.
Guo
Zhong
Z
National Chromatographic R & A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
Xu
Songyun
S
Lei
Zhengdeng
Z
Zou
Hanfa
H
Guo
Baochuan
B
D000327:Adsorption; D002614:Chelating Agents; D045304:Cytochromes c / Q000032:analysis / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods; D004800:Enzymes, Immobilized / Q000032:analysis; D007202:Indicators and Reagents; D010449:Peptide Mapping / Q000379:methods*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin / Q000737:chemistry / Q000302:isolation & purification
D002614:Chelating Agents; D004800:Enzymes, Immobilized; D007202:Indicators and Reagents; D045304:Cytochromes c; D014357:Trypsin