Software using maximum entropy (MaxEnt) analysis has been developed, and used to deconvolute complete electrospray spectra of protein mixtures. It automatically produces zero-charge mass spectra on a molecular mass scale, along with probabilistic quantification so that the reliability of features in the spectrum can be ascertained. Because maximum entropy is faithful to the experimental data, the results tend to have improved resolution and signal-to-noise ratio. This improved performance, particularly regarding resolution, is demonstrated on a haemoglobin containing two &globins separated by 12 Da at mlz 15 867 (0.08°/0). A separation of 12 Da was previously the closest at which mass measurement of two globins was practicable. Also, two hitherto unresolved P-globins from a second haemoglobin, separated by 9 Da (0.06%) were resolved by MaxEnt and their masses accurately measured. These are the first results using rigorous MaxEnt analysis in electrospray mass spectrometry.In the form initially produced by the mass spectrometer, the electrospray spectra of protein mixtures are complex, each protein in the mixture being represented by a series of multiply charged ions on a madcharge ratio scale.Generally these ions occur with mass-to-charge ratio ( M + z H ) l z , where M is the molecular mass of the protein, H' is the mass of the proton (1.00794 u) and z is the number of charges on an ion, a series of consecutive integers. A 15 kDa protein typically produces about 10 peaks with a range of z from 10-20. Larger proteins, e.g., albumins (66kDa) often have 20 or more peaks in the series.To aid interpretation, some means is required for generating a zero-charge mass spectrum, whereby each component in the mixture is transformed from the multiply charged ion series on a masslcharge ratio scale to a single peak on a molecular mass scale. Early methods] tended to produce artefacts and a baseline which increased with mass. A more recent approach2 has achieved an artefact-free zero-charge spectrum with an improved signal-to-noise ratio, but requires prior identification of the charge states in the multiply charged ion series. Current software allows semiautomatic identification of the charge states, but nevertheless a degree of operator intervention is usually required. Moreover, since the data in the original multiply charged spectrum are directly transformed onto a true molecular mass scale, components which are unresolved in the original data remain unresolved after transformation to the zero-charge spectrum. Yet the signals are known to be broadened, by both the isotopic distribution of the elements in the molecule (isotopic broadening) and the mass spectrometer. Hence the true underlying spectrum of masses will be sharper than the peaks in the original data. By incorAuthor to whom correspondence should be addressed. porating this broadening into the program, MaxEnt is able to deconvolute it from the data, thus enhancing the resolution which can be observed in the final MaxEnt mass spectrum. Peak heights grow proportiona...
A mechanism for the production of multiply charged molecular ion species in electrospray mass spectrometry (ES-MS) is still required. A concise discussion of a recently published ionic solution equilibrium model offering a partial mechanism is presented. That publication proposed that the ion abundance-charge profile could be fitted by one or a series of superimposed Gaussian functions, in accord with a solution equilibrium model. It is shown that indeed a simulated mass spectrum based on a solution model can compare well with the observed spectrum. However, some new and recently published experimental evidence is presented which shows that the ES mass spectra of many proteins give rise to multiply charged molecular ions which carry higher charges than those calculated by the model. Further, the ion abundance-charge profile is very sensitive to some experimental parameters, e.g. cone voltage; it does not necessarily reflect the solution or gaseous ion populations in the mass spectrometer source. Therefore, the concept that gaseous multicharged ions originate from equivalently charged solvated ions in electrically neutral solution must be treated with caution.
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