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Novel methods to study protein dynamics using electrospray ionization mass spectrometry
Protein ion charge state distributions in Electrospray ionization mass spectrometry (ESI MS) provide information on protein dynamics, as it contains contributions from all protein conformers present in solution. Such ionic contributions often overlap, limiting the amount of useful information that can be extracted from the spectra. This difficulty is overcome by using factor analysis, which allows spectral deconvolution to be carried out and information on individual protein conformers to be extracted. Experiments were carried out by acquiring a series of spectra over a range of near-native and denaturing conditions and then subjecting to singular value decomposition, to determine the number of conformers. Ionic contributions of each conformer to the total signal are then determined using a supervised minimization routine. By monitoring acid and alcohol induced equilibrium intermediates of well-characterized model proteins we can demonstrate that factor analysis correctly predicts the total number of conformations of the model proteins under a variety of conditions. Several factors have also been suggested as determinants of the number of charges displayed by the protein ions in electrospray ionization (ESI) spectra, such as available basic residues, solvent composition, etc. This work demonstrates that protein surface area in solution is the major determinant of the number of charges accommodated by a protein ion in the gas phase. The charged residue model of ESI process predicts that the number of charges (N) accommodated by a globular protein in the gas phase should be related to its surface area (S) as N∼S3/4. We analyzed a set of proteins ranging from 5 kDa insulin to 0.5 MDa, ferritin under native conditions. The average charge of each protein was plotted as a function of its surface area (calculated based on the available crystal structures), giving an apparent correlation of N∼S0.68. Effects of gas phase chemistry on the charge state distribution was investigated. It is seen that using a stronger base as a electrolyte component resulted in a noticeable decrease of the average charge, without affecting the correlation N∼S 0.68. At high pH charge state distribution is shifted to lower charges, attributed to the free base in solution. ^
"Novel methods to study protein dynamics using electrospray ionization mass spectrometry"
(January 1, 2005).
Electronic Doctoral Dissertations for UMass Amherst.