New ultrasonic-based methods in proteomics

Dissertação apresentada para obtenção do Grau de Doutor em Bioquímica pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia.A presente dissertação foi preparada no âmbito do convénio bilateral existente entre a Universidade Nova de Lisboa e a Universidade de Vigo.

The present dissertation is devoted, essentially, to the development of new ultrasonic-based sample preparation methods for mass-spectrometry-based analysis of proteins. This dissertation is introduced by a brief overview about general aspects in proteomics such as protein identification, quantification and finally, tissue analysis by mass spectrometry. Special attention is given to fast methods for protein identification, as for example those using tools such as microwave energy and ultrasonic energy to speed-up enzymatic digestion. In addition, a general overview of label-based quantification approaches, such as SILAC, iTRAQ, ICAT, or 18O and, label-free methods are presented. Furthermore, stable 18O-isotopic labeling is described in detail. The last topic covered in the first chapter is the analysis of tissue samples by mass spectrometry, a short overview on this subject is done to introduce the reader on the importance of tissue analysis by mass spectrometry. As far as experimental concerns, different types of ultrasonic devices, such as ultrasonic bath, sonoreactor, ultrasonic probe and multiprobe were assessed in different steps of currents protocols for protein identification through mass spectrometry. Variables such as temperature, sonication time, amplitude and ultrasonic frequency were evaluated in order to identify the optimum conditions. On the first trial, ultrasonic energy was used to accelerate the in-solution enzymatic protein digestion. It was found that the application of the ultrasonic energy (ultrasonic probe and sonoreactor) to a liquid medium containing protein and trypsin speeds protein cleavage from overnight (12h) to 5min. To make the handling easier and faster, the ultrasonic energy was also successfully applied to the protein reduction and protein alkylation steps. Overall, the time was reduced from 3h to 10min, and from many single cleaning steps to just one for reduction and another for alkylation.