Development of new methodologies in sample treatment for proteomics workflow based on enzymatic probe sonication technology and mass spectrometry

Thesis submitted to the Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia, for the degree of Doctor of Philosophy in Biochemistry

In 1994, Marc Wilkins coined the word “Proteome” to define the protein product of the genome, and the word “Proteomics” to describe the science that studies the proteome. Since then, boosted by the development of soft ionization technologies, mass spectrometry and analytical and bioinformatic tools, proteomics has become one of most important and popular scientific fields for the large scale study of complex protein systems. Nowadays, the wide range of proteomics applications include not only the study of biological functions, but also the understanding of changes in cellular regulation mechanisms caused by disease states, biomarker identification for disease diagnosis and development of new drugs or therapeutic approaches. To achieve these goals, most proteomics studies rely on different but complementary tools, such as: two-dimensional gel electrophoresis, chromatographic separation methods, mass spectrometry, stable isotope labeling approaches for protein quantitation, and software for data collection and analysis. However, despite the many technological advances, the procedures used for protein identification and quantitation are still complex, lengthy and laborious. One of the most important steps in any protein identification or quantitation experiment is the digestion, or hydrolysis, of proteins. This crucial step is traditionally performed with proteases, such as trypsin, during 12 to 48 h. Over the years, many techniques have been used to optimize the sample treatment procedures in proteomics, particularly the protein enzymatic digestion stage. The most popular are microwave energy, high-pressure reactors, micro-reactors and immobilized enzymes. In 2005, ultrasonic energy was used for the first time to enhance protein enzymatic digestion in proteomics workflow. Promising results were obtained: the protein digestion time was reduced from 12 h to only 120 s. Yet, many aspects regarding the application of the ultrasonic energy to the digestion of proteins with enzymes still remain unclear and not fully comprehended. The major objective of this dissertation was the development and optimization of protocols relying on ultrasonic energy to enhance protein identification and quantitation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). With this intention, different ultrasonic devices, such as the ultrasonic bath, the ultrasonic probe and the sonoreactor, were used to enhance several steps of the traditional procedures for protein identification by peptide mass fingerprinting (PMF), and protein quantitation by 18O isotopic labeling. Among the different variables assessed throughout this work, the most important were: (i) ultrasound amplitude; (ii) ultrasonication volume; (iii) ultrasonication time; (iv) ultrasound frequency; (v) ultrasonic probe diameter; (vi) protein concentration threshold; and (vii) enzyme-to-protein ratio.In the first part of this study, the experiments were focused on the application of ultrasonic energy to the in-gel protein enzymatic digestion for PMF identification. Different proteins were separated by one-dimensional gel electrophoresis and the corresponding gel bands were processed according to the already established procedures. Protein digestion with trypsin was carried out with different ultrasonic devices: the ultrasonic probe and the sonoreactor.

Fundação para a Ciência e a Tecnologia - SFRH/BD/28563/2006