In
ESI
or
MALDI, spectra often only
contain the ionised molecule with very little fragmentation data and
consequently the spectra are of little use for structural
characterisation. In the cases when structural data is required,
tandem mass spectrometry (MS/MS) can be
routinely employed. Unfortunately, though, there are many occasions
when MS/MS either doesn't provide enough fragmentation information
(especially for some types of analyte that only loose simple neutrals
in MS/MS) or provides a lot of confusing information (especially when
the analyte fragments by multiple pathways). In both of these cases,
multistage tandem mass spectrometry is required (MS
n).
MS
n is a technique, that can only be
performed on
ion-trap and
FT-ICR instruments, which allows the
re-fragmentation of product ions (fragment ions from MS/MS). Figure 1
shows a schematic of the MS
n experiment. In the first stage,
the normal mass spectrum is produced in the usual way. The isolation of
the precursor ion is then performed which is fragmented by CID in
exactly the same way is in MS/MS. In MS/MS, the experiment would end
here as the product ions are consumed by the detector, in MS
n
though, the product ions are trapped allowing another isolation and
fragmentation to be performed resulting in the MS
3 spectrum.
This process can be repeated a number of times, resulting is a series
of MS
n spectra where 'n' represents the number of times the
isolation-fragmentation cycle has been carried out.
Figure 1: A Schematic of
multistage tandem mass spectrometry.
Figure 2 shows a
fictional example of an MS3 experiment. In the normal mass
spectrum, the [M+H]+ ion is the base peak, with a series of
other ions due to impurities or other components of the analyte
solution. These 'unwanted' ions are removed by the isolation step (it
is also possible in FT-ICR-MSn to remove the isotopes of the
precursor ion leading to isotopically pure product ion spectra) and the
precursor ion subjected to CID fragmentation. The resultant MS2
spectrum (the product ion spectrum) now shows a small precursor ion and
a series of fragment ions. In the example, two of these fragment ions
are re-isolated and fragmented to produce two MS3 spectra
showing sub-fragmentation. This allows for relationships between
product ions and precursor ions to be established and for the
separation of multiple fragmentation routes. Also, if the analyte does
not fragment very well in MS/MS (is very stable) re-fragmentation of a
water loss peak (for example) will often produce a very good set of
fragment ions allowing structural data to be obtained which would
otherwise be lost with MS/MS analysis only.
Figure 2: A fictional example of multistage mass spectrometry (in this
case MS
3).
©2004 Paul
Gates, University of Bristol
Last updated February 23rd 2004
