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...

The maximum entropy (MaxEnt) technique has been well established in image processing for the last decade. In more recent times MaxEnt has been applied to spectroscopic techniques and shows considerable promise. As part of a much wider evaluation of the Cambridge software, specifically MemSys3, the MaxEnt technique has been successfully applied to electrospray mass spectra.

The applicability of coupled reversed-phase high performance liquid chromatography (HPLC)-NMR spectroscopy for the detection and identification of paracetamol (N-(4-hydroxyphenyl)acetamide) and its sulfate, glucuronide and N-acetylcysteinyl metabolites in the unprocessed biological fluids, human urine, rat urine and rat bile, is investigated. Analysis of these samples was performed by gradient HPLC elution and directly coupled 500 MHz 1H NMR spectroscopy detection using a combination of one- and two-dimensional NMR methods in stopped-flow mode. The stopped-flow approach is demonstrated to be an efficient technique for identification of drug metabolites which have, for example, a UV-chromophore. Stopped-flow HPLC analysis with NMR detection is a viable technique and halting the chromatographic process several times during a run has a negligible effect on the separation and NMR characterization. The post-acquisition data processing method of 'quantified maximum entropy' is shown to provide a means of improving the quality of spectra for minor components, thus aiding NMR resonance assignments.

The MaxEnt technique has premviously been successfully applied to the deconvolution of electrossppray mass spectra. The latest version of the Cambridge University software, ‘MemSys5’, has now been applied to high resolution mass spectra. Initial results have shown that peaks requiring an instrument resolution of almost 200 000 to separate them are readily resolved by MaxEnt on data acquired at a resolution of only 50, 000. Moreover, the MaxEnt results are accompanied by quantitative and realistic error bars.

The MaxEnt technique has previously been successfully applied to the deconvolution of electrospray mass spectra. The latest version of the Cambridge University software, 'MemSysS', has now been applied to high resolution mass spectra. Initial results have shown that peaks requiring an instrument resolution of almost 200000 to separate them are readily resolved by MaxEnt on data acquired at a resolution of only 50000.Moreover, the MaxEnt results are accompanied by quantitative and realistic error bars.