GOMEZ Gabriela Elena
congresos y reuniones científicas
Probing Protein Conformation with a General Probe Coupled to Detection by Mass Spectrometry
Boston, USA
Simposio; Nineteenth Symposium of The Protein Society.; 2005
Probing Protein Conformation with a General Probe Coupled to Detection by Mass Spectrometry G¨®mez, G.E., Delfino, J.M. IQUIFIB (Facultad de Farmacia y Bioqu¨ªmica, Universidad de Buenos Aires-CONICET), Jun¨ªn 956, (C1113AAD), Buenos Aires, Argentina. E-mails:, Diazirine (DZN) is a photoreactive gas similar in size to water. Upon photolysis it generates methylene carbene (:CH2), which reacts unselectively inserting even into C-H bonds. 3H-DZN was used in our laboratory for studying protein folding and for mapping the area of interaction in protein complexes. Methylation is unspecific and depends primarily on the solvent accessible surface area (SASA) of the polypeptide chain. So far 3H-DZN proved to accurately quantify the extent of modification even at very low levels. We investigated the feasibility of a non-radioactive methylene labeling procedure coupled to detection by mass spectrometry (ES-MS), thus avoiding handling of radiotracers. Such analysis let us to reduce the needed amount of sample and opens the possibility of automation. To achieve these goals we developed a device which takes advantage of the continuous dissolution and photolysis of DZN into the sample enhancing the labeling yield. In a first test reaction run with hen egg white lysozyme the resolution of MS spectra allowed us to clearly distinguish M + n*14 peaks, as well as to evaluate the increase in the intensity of those peaks in the unfolded state U relative to a sample modified in the native state N. Here we assess in a quantitative fashion the conformational state of another model protein: bovine ¦Á lactalbumin (¦ÁL¦¡). The urea-induced unfolding transition of ¦ÁLA monitored by the extent of methylation agrees well with measurements by circular dichroism spectroscopy in both ultraviolet regions. The modification ratio U/N can be related to the increment expected from theoretical estimates of SASA in state U. This method -which relies on a novel application of MS to the detection of methylated products- allows a straightforward measurement of SASA and proves to be sensitive to conformational changes of the polypeptide chain. Supported by CONICET, ANPCyT and UBA.