GOMEZ Gabriela Elena
congresos y reuniones científicas
Labeling of Proteins with the Minimal Probe Diazirine Coupled to Detection by Mass Spectrometry.
Rosario, Santa Fe, Argentina
Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica; 2006
Diazirine (DZN) is a photoreactive gas similar in size to water. By UV irradiation it generates methylene carbene (:CH2), which reacts unselectively with its molecular cage, inserting even into C-H bonds. 3H-DZN has been successfully used in our laboratory for studying protein structure and folding [1, 2] and for mapping the area of interaction in protein complexes [3]. The labeling phenomenon (methylation) is unspecific and depends primarily on the solvent accessibility of the polypeptide chain. This method addresses experimentally the measurement of the solvent accessible surface area (SASA) in biomolecules. So far 3H-DZN proved to be useful to accurately quantitate the extent of modification even at very low levels. However, this requires the handling of expensive radiotracers. In this context, we investigated the feasibility of a general labeling procedure based on non-radioactive methylene coupled to detection by mass spectrometry (ES-MS). To achieve these goals we developed a device that takes advantage of the continuous dissolution and photolysis of DZN into the sample, thus enhancing the labeling yield. Due the high resolution, peaks (M + n*14) are clearly distinguishable in the ES-MS spectra. By evaluating their position and intensity, one can derive an estimate of the extent of modification with methylene (EMM: measured as moles of :CH2 incorporated per mole of protein at a given DZN concentration). A linear dependence of EMM with DZN concentration was observed, in a fashion similar to measurements carried out before with 3H-DZN. Labeling of globular proteins (ubiquitin, a-lactalbumin, hen egg white lysozyme, TPCK treated trypsin, a-chymotrypsin, subtilisin Carlsberg and B. licheniformis b-lactamase) in their native or unfolded states systematically shows a higher EMM in the latter case, as expected from the predicted SASA increment occurring upon unfolding. Furthermore, we investigated in detail the urea-induced conformational transition experienced by a-lactalbumin. Here, the EMM monitored along this transition superimposes over measurements derived from circular dichroism in both far and near ultraviolet regions, and intrinsic fluorescence emission. This development represents a novel application of MS to the conformational analysis of proteins. Supported by Fundación YPF, CONICET, UBA and ANPCyT. References: [1] Patricio O Craig, Daniela B Ureta, José M Delfino, Protein Science, 2002, 11, 1353-1366. [2] Daniela B Ureta, Patricio O Craig, José M Delfino, Protein Science, 2001, 10, suppl 1, 131. [3] Gabriela E. Gómez, Ana Cauerhff, Patricio O. Craig, Fernando A. Goldbaum, and José M. Delfino, Protein Science, 2006, 15, 744-752.