Patrik Vrábel

Study of Fluorescently Labelled Peptides and Small Proteins Using Capillary Electrophoresis Combined with Laser Induced Fluorescence and MALDI MS Detection

In this work, we apply CE-LIF, CE-UV and CE - Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI TOFMS) to investigate derivatization of model peptides and small proteins with rhodamine-based fluorescent reagents, which are compatible with the wavelength of frequency doubled diode pumped Nd:YAG excitation laser (532 nm). While the LIF detection mode is needed for sensitive detection, the MALDI TOFMS analysis can reveal the mass of analytes. Both derivatized and non-derivatized peptides and proteins, the number of fluorescent labels per peptide/proteins and other product of derivatization process can be detected directly from reaction mixtures or after separation. Thus, MALDI TOFMS serves for additional identification of peaks present in CELIF or CEUV electropherograms. For off-line coupling of CE with MALDI MS, subatmospheric deposition interface was used. An examples of final sensitive CELIF separation of derivatized peptides and small proteins are given. Main advantages and disadvantages of LIF, absorbance and MS detector for capillary electrophoresis of polypeptides are disscused. Attention is paid to detection limits in practical analysis.