Monitoring the changes in charge-state distributions of protein ions in electrospray ionization (ESI) mass spectra has become one of the commonly accepted tools to detect large-scale conformational changes of proteins in solution. However, these experiments produce only qualitative, low-resolution information. Our goal is to develop a procedure that would produce quantitative data on protein conformational isomers coexisting in solution at equilibrium. To that end, we have examined the evolution of positive ion charge-state distributions in the

Protein ion charge state distributions in electrospray ionization mass spectra have a potential to provide a wealth of information on protein dynamics, because they contain 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 in the present work by using a chemometric approach, which allows spectral deconvolution to be carried out and information on individual protein conformers to be extracted. Experiments are carried out by acquiring a series of spectra over a range of near-native and denaturing conditions to ensure significant changes in the conformers' populations. A total number of protein conformers sampled in the course of the experiments is determined by subjecting the set of collected spectra to singular value decomposition. Ionic contributions of each conformer to the total signal are then determined using a supervised optimization routine. Validation of the method has been carried out by monitoring acid- and alcohol-induced equilibrium states of well-characterized model proteins, chymotrypsin inhibitor 2 (two states), ubiquitin (three states) and apo-myoglobin (four states). For each of the model proteins, a new chemometric procedure yielded a picture of protein dynamics that was in excellent agreement with their documented behavior (as studied with other biophysical tools). The new method appears to be well-suited for monitoring protein dynamics in highly heterogeneous systems consisting of multiple proteins sampling a range of conformational states.

Electrospray ionization mass spectrometry (ESI-MS) is often used to monitor protein conformational dynamics in solution, for example acid unfolding, by following the changes in positive-ion charge-state distributions in response to changes of ambient conditions, for example solution pH. Deconvolution of these charge-state distributions often reveals the presence of multiple protein conformers coexisting in solution in equilibrium. The ion signal corresponding to each conformer depends on its size (which determines the average charge state of the protein ions) and heterogeneity (which determines the spread of the ion signal). In the present work, we seek to explore how the ion signal of individual protein conformers can be influenced by other factors not related to protein shape, with particular attention being paid to contributions from solution acid-base chemistry. The composition of the buffer was found to exert a significant influence on the ion signal by inducing apparent charge reduction of the protein ions. This effect was ascribed to protein-base (anion) complex formation in solution followed by dissociation of the neutral conjugated acid from the complex in the gas-phase. The resulting shift in the charge-state distribution occurs in the pH range from p Ka to approximately (p Ka −1.5) and is induced by the elevated concentration of the anion in solution. On the other hand, intrinsic charges on the protein in solution have been shown to have no effect on the appearance of the charge-state distributions, lending further credibility to the notion that protein shape is the only structural determinant of the ion signal in ESI-MS.

Different types of cyclic phosphine oxides, such as tetrahydrophosphole oxide 1, phosphabicyclo[3.1.0]hexane 3oxide 8 and phosphabicyclo[2.2.1]heptene 7-oxides 10 and 12 were efficiently converted to phosphine-boranes 2, 9, 11 and 13, respectively, under relatively mild conditions by reaction with 4.4 equivalents of dimethyl sulfide-borane. The more strained hetero-ring the starting phosphine oxide (in general 16) has, the easier to accomplish the change in the P-function, that takes place through the corresponding phosphine intermediate ( 20). It is noteworthy that the imide carbonyl groups in starting materials 10 and 12 were fully reduced by the borane to give 11 and 13 respectively.

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