Open-tubular capillary electrochromatography coupled with electrospray ionization mass spectrometry for peptide analysis.
In this study, the open-tubular electrochromatographic (OT-CEC) migration behavior of various peptides has been characterized using etched and chemically (n-octadecyl- and cholesterol-) modified capillaries, interfaced to an electrospray ionization mass spectrometer through a sheath liquid configuration. The stationary phases were fabricated by etching the inner wall of the fused-silica capillary and then chemically modifying the new surface through a silanization/hydrosilation reaction. Unlike some other OT-CEC stationary-phase preparation methods, leaching of the immobilized stationary phase and subsequent contamination of the electrospray ion source was largely avoided with this novel surface modification technology. The influence of the immobilized organic phases and those of the buffer electrolytes (pH, the type and content of organic solvent) on the retention and separation of the selected peptides was investigated. Significant peptide retention was found even at very low pH with both types of stationary phases, under conditions whereby the electrophoretic migration dominated the separation process. Due to the effective coverage of the etched surface by a silanization/hydrosilation reaction, adverse adsorption of charged analytes onto the capillary wall was minimized. As a result, very efficient and highly reproducible peptide separations were achieved over a broad pH range. Moreover, peptide-specific multizoning effects were observed. The origin of this novel phenomenon was explored. Compared to capillary electrophoresis electrospray ionization mass spectrometry system, much higher detection sensitivity could be obtained, since a larger amount of sample could be injected and stacked at the head of the open-tubular capillary column without deteriorating the separation performance. On the basis of these observations, these procedures have been adapted to allow the analysis of tryptic peptides generated from proteins.
Yang
Yuanzhong
Y
Australian Research Council Special Research Centre for Green Chemistry, Clayton, Victoria, 3800, Australia.
Boysen
Reinhard I
RI
Matyska
Maria T
MT
Pesek
Joseph J
JJ
Hearn
Milton T W
MT
D000473:Alkanes / Q000737:chemistry; D053801:Capillary Electrochromatography / Q000379:methods*; D002784:Cholesterol / Q000031:analogs & derivatives; D019075:Electrophoresis, Capillary / Q000379:methods; D006863:Hydrogen-Ion Concentration; D009930:Organic Chemicals / Q000737:chemistry; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results; D012821:Silanes / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
D000473:Alkanes; D009930:Organic Chemicals; D010455:Peptides; D012821:Silanes; D012997:Solvents; D012822:Silicon Dioxide; D002784:Cholesterol; D014357:Trypsin; C022883:octadecane