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More Information on ESMS
Impact of Multiply Charged Ions on Mass Spectrometry
Although we sometimes see +2 charged species during MALDI-MS, short peptides tend
primarily to give just the +1 species so this makes it easy to directly interpret
MALDI-MS spectra. The problem is that all types of MS actually measure the mass/charge
ratio (m/z) as opposed to the mass. Hence the following two scenarios give
identical spectra with a single observed peak (in positive ion mode) at m/z of
2,001:
Actual (M) Peptide mass = 2,000, Charge = +1, Observed (M+H) m/z =
(2,000 + 1)/1 = 2,001
Actual (M) Peptide mass = 4,000, Charge = +2, Observed (M+H) m/z = (4,000 +
2)/2 = 2,001
Multiple charge states are potentially a severe
problem for electrospray
ionization mass spectrometers. Instead of the single (M+H)
species characteristic of linear MALDI-MS, electrospray usually gives a broad spectrum of
multiply charged ions for each species present, which is why a mass spectrometer equipped
with an electrospray source that has an upper m/z limit of 1,800 can easily
determine the m/z for a 50,000 dalton protein. Because of multiple charging many m/z
ratios obtained from the electrospray ionization mass spectrometers do not correspond with
actual peptide masses.
Electrospray Data Processing on the Q-Tof Mass Spectrometer
There are two general methods for processing electrospray mass spectra into their
singly charged format on the Q-Tof mass spectrometer, namely, the transform method and the
Maximum Entropy method. The method selected depends on the quality of the raw data (e.g.,
signal to noise ratio and the number of components present in the sample). The transform
method is the preferred method if the component peaks in the multiply charged raw spectrum
can be identified. This method provides multiple measurements of the molecular weight
because each multiply charged ion is an independent measurement of the molecular weight.
From these multiple measurements an average mass can be calculated and a standard
deviation. These multi-charged spectra can then be transformed into the equivalent singly
charged spectrum. In this instance, the sample submitter would receive three types of
data, the list of the multi-charged masses and the calculated molecular weight, a plot of
the transformed, singly charged spectrum, and a plot of the multi-charged spectrum. The
major limitation of this method is the appearance of artifact peaks in the transformed
spectrum from background ions. These are usually readily apparent however because they do
not have other related component peaks.
Maximum Entropy processing is the only method that can be used to process multiply
charged spectra when adjacent component peaks cannot be identified because of low signal
or because multiple components in the sample hinder the identification. The only required
input for Maximum Entropy processing is the expected molecular mass range of the compound
of interest and an estimate of the peak width for a compound of that mass range with the
number of charges needed to produce the observed multi-charged spectrum. This latter
parameter takes into account the width of the isotope envelope and instrument resolution.
The output from a maximum entropy processed spectrum is a plot showing the singly charged
molecular ion and a reconstructed multi-charged spectrum from MaxEnt which can be compared
to the multi-charged raw spectrum which then provides a measure of the quality of the
input parameters. When MaxEnt is used sample submitters would receive copies of all three
outputs.
Spectra processed with the transform algorithm have in addition to the list of masses,
the letters Tr in the second line in the upper left hand corner of the molecular mass
spectrum. Maxent spectra contain in addition to the reconstructed multicharged spectrum
the letters Mk in the second line of the molecular mass spectrum.
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