De novo Sequencing of Proteins With Mass Spectrometry
77
have been generated from the complete tandem MS spectrum alone because too
many other ions mimic the typical 1:1 16 0/ 18 0 isotopic ratio.
Y ions can be made visible but, on the other hand, intensive unlabelled fragment ions may still be retained in the subtracted spectrum because of imperfect
matching of the two spectra. The character of the ion, however, becomes obvious
when considering its normal isotopic distribution in the original spectrum (see
Fig. 5.5).
The differential scanning approach is based on the fact that the second tandem
MS spectrum is generated exclusively from ions which carry the heavier 18 0 isotope at the C-terminus. All ions with the 16 0 isotope should be effectively
excluded from the second tandem MS experiment. The natural13C and 15N isotope contribution of the peptide needs to be taken into account. Therefore, the
transmission is often shifted to ions with a higher mass value than (M+2 Da) for
the second investigation, with M being the ion mass for the first isotope of the
peptide. As a general guideline the selection is shifted by 1 Da on the m/z scale
translating into 2 Da for doubly charged and 3 Da for triply charged ions.
The shift to higher than (M+2 Da) mass ions has a side effect. Large unlabelled fragment ions are not eliminated from the subtracted spectrum when a large
peptide precursor is fragmented. For a large precursor the ion transmission for
the second experiment needs to be shifted considerably higher than to a mass of
(M+2 Da) to exclude the 13C and 15N isotopes of the 16 0 labelled peptide. Therefore, the 12C-only isotope of the 18 0 labelled peptide is excluded as well from the
second experiment. In contrast to the oxygen isotope, which is at a fixed position,
the excluded 12C isotope randomly distributes amongst all carbon atoms in the
peptide chain. For large fragments, the probabilities add up resulting in an
under-representation of the first 12C-only isotope in the fragment spectrum.
When subtracting the second spectrum from the first, the 12C-only isotope of a
large unlabelled fragment is retained (see Fig. 5.6). Fortunately, the high m/z area
of the spectrum is virtually free of chemical noise and y ions can be recognized
by their isotopic distribution in the original 16 0/ 18 0 spectrum.
5
Summary
Differential scanning is a new technique to improve de novo peptide sequencing
with tandem mass spectrometry. Y ions are filtered from the fragment spectrum
of partially C-terminally labelled peptides. The y ion filter is based on the different isotopic representation of C-terminal ions in a fragment spectrum generated
from the complete isotopic 16 0/ 18 0 distribution and a fragment spectrum of the
18 0 containing isotopes. Both tandem MS spectra can be generated with the same
overall ion transmission of the precursor selecting quadrupole. This technique
allows to use 18 0 isotopic labeling on a triple quadrupole instrument for de novo
sequencing. In combination with a quadrupole-time of flight mass spectrometer
differential scanning improves the quality and the speed with which spectra can
be read out. The technique simplifies tandem MS spectra considerably which can
improve automatic interpretation. Currently, it is preferable to determine the
amino acid sequences manually. This allows to combine information from the y
77
have been generated from the complete tandem MS spectrum alone because too
many other ions mimic the typical 1:1 16 0/ 18 0 isotopic ratio.
Y ions can be made visible but, on the other hand, intensive unlabelled fragment ions may still be retained in the subtracted spectrum because of imperfect
matching of the two spectra. The character of the ion, however, becomes obvious
when considering its normal isotopic distribution in the original spectrum (see
Fig. 5.5).
The differential scanning approach is based on the fact that the second tandem
MS spectrum is generated exclusively from ions which carry the heavier 18 0 isotope at the C-terminus. All ions with the 16 0 isotope should be effectively
excluded from the second tandem MS experiment. The natural13C and 15N isotope contribution of the peptide needs to be taken into account. Therefore, the
transmission is often shifted to ions with a higher mass value than (M+2 Da) for
the second investigation, with M being the ion mass for the first isotope of the
peptide. As a general guideline the selection is shifted by 1 Da on the m/z scale
translating into 2 Da for doubly charged and 3 Da for triply charged ions.
The shift to higher than (M+2 Da) mass ions has a side effect. Large unlabelled fragment ions are not eliminated from the subtracted spectrum when a large
peptide precursor is fragmented. For a large precursor the ion transmission for
the second experiment needs to be shifted considerably higher than to a mass of
(M+2 Da) to exclude the 13C and 15N isotopes of the 16 0 labelled peptide. Therefore, the 12C-only isotope of the 18 0 labelled peptide is excluded as well from the
second experiment. In contrast to the oxygen isotope, which is at a fixed position,
the excluded 12C isotope randomly distributes amongst all carbon atoms in the
peptide chain. For large fragments, the probabilities add up resulting in an
under-representation of the first 12C-only isotope in the fragment spectrum.
When subtracting the second spectrum from the first, the 12C-only isotope of a
large unlabelled fragment is retained (see Fig. 5.6). Fortunately, the high m/z area
of the spectrum is virtually free of chemical noise and y ions can be recognized
by their isotopic distribution in the original 16 0/ 18 0 spectrum.
5
Summary
Differential scanning is a new technique to improve de novo peptide sequencing
with tandem mass spectrometry. Y ions are filtered from the fragment spectrum
of partially C-terminally labelled peptides. The y ion filter is based on the different isotopic representation of C-terminal ions in a fragment spectrum generated
from the complete isotopic 16 0/ 18 0 distribution and a fragment spectrum of the
18 0 containing isotopes. Both tandem MS spectra can be generated with the same
overall ion transmission of the precursor selecting quadrupole. This technique
allows to use 18 0 isotopic labeling on a triple quadrupole instrument for de novo
sequencing. In combination with a quadrupole-time of flight mass spectrometer
differential scanning improves the quality and the speed with which spectra can
be read out. The technique simplifies tandem MS spectra considerably which can
improve automatic interpretation. Currently, it is preferable to determine the
amino acid sequences manually. This allows to combine information from the y