Microwave-assisted protein hydrolysis

Early studies in microwave-assisted hydrolysis of proteins showed the recoveries of amino acids after microwave hydrolysis to be quite consistent with the results provided by conventional hydrolysis [293,294]. Engelhardt et al. used on-line microwave hydrolysis in an HPLC system followed by derivatization with OPA to improve the detection sensitivity for proteins [295]. 

The initial work on microwave-assisted hydrolysis of proteins was conducted using commercial domestic microwave devices. Different laboratory microwave ovens featuring various methods of operation are now commercially available as well. The microwave-assisted hydrolysis of proteins or peptides is usually performed in sealed containers [296] where the sample is in direct contact with the concentrated acid used for hydrolysis. In vapour-gas microwave hydrolysis [297], the acid is evaporated and only the vapour contacts the sample, which prevents contamination of the samples by small amounts of amino acids potentially present in the acid used for analysis. 

Microwave-assisted hydrolysis systems equipped with temperature regulators suitable for quality control of protein products are now available. In fact, microwave hydrolysis is the sole technique in which the acid temperature and vapour pressure are measured directly, in situ. Other devices measure the temperature of a heating unit such as metal heating block or oven surrounding a hydrolysis chamber. 

One major concern in the hydrolysis process is the potential racemization of amino acids. Peter et al. [298] used both microwave and conventional hydrolysis to determine the amino acid composition of three synthetic peptides and found microwave-assisted hydrolysis to result in reduced racemization and in higher recovery of sensitive amino acids than hydrolysis by conventional heating with hydrochloric acid [299]. 

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Zhong, H., Marcus, S.L. and Li, L. (2005) Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification. J. Am. Soc. Mass Spectrom. 16, 471-481. 

A rapid microwave-assisted protein digestion techniqne based on classic acid hydrolysis reaction with 2% formic acid solution has been investigated by Hna et al. (2006). In this mild chemical environment, proteins were hydrolyzed to peptides. It was observed that dilnte formic acid cleaved proteins specifically at the C-terminal of aspartyl (Asp) residnes within 10 min of exposure to microwave irradiation. On the basis of the observations of the single fragmentation of myoglobin at the C-ter-minal of any of the Asp residnes, they found that the extent of protein fragmentation could be controlled as compared to native fluoreseenee detection. 

Before applications are dealt with, the main variables governing microwave-assisted processes and the parameters characterizing specific microwave treatments are examined. The applications discussed include not only microwave-assisted digestion and extraction — which are the two most widely implemented and hence those with the highest potential interest to readers — but also others of special significance to solid sample treatment such as microwave-assisted drying, distillation and protein hydrolysis. Finally, some safety recommendations on the use of microwave equipment are made. 

Finally, other salient uses of microwaves for treating solid samples such as microwave-assisted drying, distillation and protein hydrolysis are also briefly described. 

While free amino acids can be directly extracted from food matrices, a preliminary hydrolysis step (normally microwave-assisted to speed up the hydrolysis reaction) is necessary to liberate protein-bound amino acids. After that, a sample cleanup using a precipitation agent, ultrafiltration, or SPE on Cis cartridges is carried out to remove interferences and concentrate these compoimds. 

MR provides a very simple and effective analytical method. Hydrolysis of peptides or proteins (may be microwave assisted) followed by derivatization of the resulting amino acids with the chiral MR adds a highly absorbing chromophore that converts the amino acids into UV-active diastereomers. This allows separation of D- and L-amino acids as diastereomers in the nanomole range on a nonchiral reversed phase standard column with the inherent rapidity of determination in HPLC. In addition, these derivatives of amino acids can be detected in both, simple UV as well as more selective mass spectrometric devices. 

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