On-column tryptic mapping of proteins using metal-ion-chelated magnetic silica microspheres by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Peptide mapping analysis, utilizing an easily replaceable and regenerable on-column enzymatic microreactor with metal-ion-chelated adsorption of enzyme on magnetic silica microspheres, combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), was developed. Firstly, magnetic microspheres of small size and strong magnetism were prepared through solvothermal reaction. Thereafter, by introducing tetraethyl orthosilicate (TEOS), magnetic silica (MS) microspheres were formed. Trypsin could then be immobilized onto the MS microspheres based on the Lewis acid-base interaction through the divalent cation chelators such as iminodiacetic acid (IDA), which was chemically bound to the microspheres through the introduction of glycidoxypropyltrimethoxysilane (GLYMO). The trypsin-immobilized MS microspheres were then locally packed into the capillary by the application of a strong magnetic field using a magnet. The performance of the method was exemplified with digestion of bovine serum albumin for 5 min at 50 degrees C and the result was comparable to the 12 h in-solution digestion. The ability of regeneration of the prepared on-column microreactor and good reproducibility of microreactor before and after regeneration were also demonstrated.
Li
Yan
Y
Department of Chemistry, Fudan University, Shanghai 200433, China.
Yan
Bo
B
Xu
Xiuqing
X
Deng
Chunhui
C
Yang
Pengyuan
P
Shen
Xizhong
X
Zhang
Xiangmin
X
D000327:Adsorption; D002614:Chelating Agents / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry; D008280:Magnetics; D008863:Microspheres; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry*
D002614:Chelating Agents; D004800:Enzymes, Immobilized; D011506:Proteins; D012822:Silicon Dioxide; D014357:Trypsin