Capillary electrophoresis mass spectrometry coupling with immobilized enzyme electrospray capillaries.
Open tubular capillary enzyme reactors were studied for rapid protein digestion and possible on-line integration into a CE/ESI/MS system. The need to minimize the time of the analyte molecules to diffuse towards the surface immobilized enzyme and to maximize the surface-to-volume (S/V) ratio of the open tubular reactors dictated the use of very narrow bore capillaries. Extremely small protein amounts (atto-femtomoles loaded) could be digested with enzymes immobilized directly on the inside wall of a 10 microm I.D. capillary. Covalently immobilized L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-trypsin and pepsin A were tested for the surface immobilization. The enzymatic activity was characterized in the flow-through mode with on-line coupling to electrospray ionization-time of flight-mass spectrometer (ESI/TOF-MS) under a range of protein concentrations, buffer pH's, temperatures and reaction times. The optimized reactors were tested as the nanospray needles for fast identification of proteins using CE-ESI/TOF-MS.
Krenková
Jana
J
Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Veverí 97, 602 00 Brno, Czech Republic.
Klepárník
Karel
K
Foret
Frantisek
F
D020099:Coated Materials, Biocompatible; D004573:Electrolytes; D019075:Electrophoresis, Capillary / Q000379:methods*; D046209:Electrophoresis, Microchip; D004800:Enzymes, Immobilized / Q000138:chemical synthesis* / Q000145:classification; D005240:Feasibility Studies; D008832:Microchemistry; D009862:Online Systems; D010434:Pepsin A / Q000378:metabolism; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D020099:Coated Materials, Biocompatible; D004573:Electrolytes; D004800:Enzymes, Immobilized; D011506:Proteins; D014357:Trypsin; D010434:Pepsin A