Matrix assisted laser desorption ionization for characterization of  higher molecular thiols in biological samples

Merlos Rodrigo, Miguel Ángel

Among the heavy metal-binding ligands in cells the phytochelatins (PCs) and metallothioneins (MTs) are the best characterized. PCs and MTs are different classes of cysteine-rich, heavy metal-binding molecules. PCs are enzymatically synthesized peptides, whereas MTs are gene-encoded polypeptides. Several analytical methods have been used to analyze PCs, MTs and complexes of metal in different studies. Today, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) is renowned for its extreme ease of operation and requirement of inexpensive matrices for preparation of sample; more importantly, the instrumentation is capable to be fully automated. In this study, the optimal condition for the characterization of PC2 and Cd-PC2 complex by MALDI-TOF MS was determined. The human MT2A and MT3 genes were expressed in heterologous organisms (Escherichia coli) by cloning. Further, MT2 protein was isolated from rabbit liver. MTs were purified by fast protein liquid chromatography (FPLC). The present study demonstrates analytical approaches of employing MALDI-TOF MS for characterization MTs. Other electrochemical methods were used for confirmation of thiols complexes with metal. Moreover, easy-touse instruments for MTs isolation, coupled with simple analytical detection method in fully automatic mode, providing prototype for the construction of sensor was designed

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