Disentangling electrospray spectra with maximum entropy

Abstract

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...

Probing noncovalent protein—Ligand interactions of the cGMP-dependent protein kinase using electrospray ionization time of flight mass spectrometry

“…Addition of free cGMP to the electrospray solution at an estimated cGMP concentration of 3 M results in a direct mass shift of the ion signals of PKG as illustrated for [M ϩ 25H] 25ϩ ion signal shown in Figure 2a. Deconvolution [19,20] (MaxEnt1) of the charge state envelope indicates that the mass of PKG has increased with ϳ700 Da (Figure 2b), which would correspond to the binding of two cGMP molecules (M r cGMP ϭ 345.2). Increasing the amount of cGMP added to 10 M results in a further shift of the ion-signals to higher m/z ratios.…”

“…The mass spectrometer was calibrated on the singly charged Cs nϩ1 I n clusters obtained after electrospraying an aqueous cesium iodide solution (1 mg/ml). Molecular masses of protein and protein-ligand complexes were calculated using a maximum entropy (MaxEnt1) based approach [19,20] incorporated as part of the MassLynx software (MassLynx version 3.5) supplied with the mass spectrometer.…”

The use of mass spectrometry to examine the formation and hydrolysis of the phosphorylated form of phosphoglycerate mutase

“…The MaxEnt deconvolution procedure [13] was applied for quantitative analysis of the raw data using 1.0 Da peak width and 1 Da/channel resolution.…”

Acyl Carrier Protein Is a Cellular Target for the Antibacterial Action of the Pantothenamide Class of Pantothenate Antimetabolites

“…Data were collected for an m/z range of 500 -2500 at a cone voltage of ϩ35 V and a manual pusher time of 70 s. All other instrument settings were those typically used for protein measurements on this instrument. Deconvolution of the protein spectrum was accomplished using the maximum entropy algorithm of the MassLynx software (Micromass Inc.) (22).…”