Near infrared spectroscopy compared to liquid chromatography coupled to mass spectrometry and capillary electrophoresis as a detection tool for peptide reaction monitoring.
Peptide interaction is normally monitored by liquid chromatography (LC), liquid chromatography coupled to mass spectrometry (LC-MS), mass spectrometric (MS) methods such as MALDI-TOF/MS or capillary electrophoresis (CE). These analytical techniques need to apply either high pressure or high voltages, which can cause cleavage of newly formed bondages. Therefore, near infrared reflectance spectroscopy (NIRS) is presented as a rapid alternative to monitor the interaction of glutathione and oxytocin, simulating physiological conditions. Thereby, glutathione can act as a nucleophile with oxytocin forming four new conjugates via a disulphide bondage. Liquid chromatography coupled to UV (LC-UV) and mass spectrometry via an electrospray ionisation interface (LC-ESI-MS) resulted in a 82% and a 78% degradation of oxytocin at pH 3 and a 5% and a 7% degradation at pH 6.5. Capillary electrophoresis employing UV-detection (CE-UV) showed a 44% degradation of oxytocin. LC and CE in addition to the NIRS are found to be authentic tools for quantitative analysis. Nevertheless, NIRS proved to be highly suitable for the detection of newly formed conjugates after separating them on a thin layer chromatography (TLC) plate. The recorded fingerprint in the near infrared region allows for a selective distinct qualitative identification of conjugates without the need for expensive instrumentation such as quadrupole or MALDI-TOF mass spectrometers. The performance of the established NIRS method is compared to LC and CE; its advantages are discussed in detail.
Petter
Christine H
CH
Spectroscopy Group Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 52a, 6020 Innsbruck, Austria.
Heigl
Nico
N
Bachmann
Stefan
S
Huck-Pezzei
Verena A C
VA
Najam-Ul-Haq
Muhammad
M
Bakry
Rania
R
Bernkop-Schnürch
Andreas
A
Bonn
Günther K
GK
Huck
Christian W
CW
D000596:Amino Acids / Q000737:chemistry; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods; D005978:Glutathione / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D010121:Oxytocin / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D019265:Spectroscopy, Near-Infrared / Q000379:methods*; D013997:Time Factors
D000596:Amino Acids; D010455:Peptides; D010121:Oxytocin; D005978:Glutathione