72
M. WILM et aI.
ions will be represented differently-in the first spectrum by both oxygen isotopes
and in the second only by the 18 0 isotope. The second spectrum can be subtracted from the first after having scaled them to the same maxima. This generates a y ion filtered spectrum. Only the 160-isotope of y ions should be retained
in the subtracted spectrum. The limited mass resolution achievable with a triple
quadrupole instrument when used in a high transmission mode limits the quality
of the filter. We interpret spectra acquired with the differential scanning technique by using the overlaid original data. The subtracted spectrum guides the
attention more rapidly to putative y ions. Y ion only spectra are much simpler to
interpret than complete tandem MS spectra. They can in principle be generated
by an orifice fragmentation process followed by a precursor ion scan for the arginine or lysine yl ion (LehmannWD 1998). However, the double fragmentation
process limits the sensitivity of this analysis and it is not possible to work on peptide mixtures.
The differential scanning technique relies on the precise control of the ions
transmitted into the collision zone. Therefore, it is mandatory to have the first
quadrupole Ql correctly calibrated and to adjust the Ql resolution such that the
window of transmission covers a range of approximately 3 Da on the m/z scale.
The 16 0 isotope of the peptides should be correctly assigned in the Ql spectrum.
We always acquire a well resolved Ql spectrum of the peptide mixture before the
tandem MS analysis begins.
It is more difficult to identify y ions with the 33 % 18 0 labeling technique using
differential scanning than when the peptides are methylated. But since the oxygen labelling method is so much easier to use the majority of proteins sequenced
de novo in our laboratory on the triple quadrupole machine are analyzed in this
way.
4.2
Differential Scanning Based de Novo Protein Sequencing
with a Quadrupole-Time of Flight Mass Spectrometer
The advantage of the differential scanning technique is clearly visible for spectra
generated with a quadrupole-time of flight instrument. The high resolution of
the spectra allows to use 50 % 18 0 labeling. The instrument parameters can be
adjusted by observing the isotopes transmitted into the collision cell with a low
collision energy setting before the tandem MS investigation starts.
Subtraction of the 18 0 tandem mass spectrum from the 16 0/ 18 0 spectrum can
yield a clear y ions only spectrum (see Fig. 5.3). The subtracted spectrum has a
far lower complexity compared to the original one. Because of its simplicity and
the high mass accuracy, there is often only one possibility to assemble larger ions
into a series of amino acids. The filtered spectrum can serve as a source of information to interpret tandem MS spectra automatically. The original spectra contain more fragment ions like a, b and internal fragment ions but the subtracted
spectrum helps in understanding the complete fragmentation pattern due to its
relative simplicity. About 50 % of the peptides investigated so far using this technique could be correctly interpreted automatically from the subtracted spectrum
alone. When interpreting the original data together with the subtracted spectrum
M. WILM et aI.
ions will be represented differently-in the first spectrum by both oxygen isotopes
and in the second only by the 18 0 isotope. The second spectrum can be subtracted from the first after having scaled them to the same maxima. This generates a y ion filtered spectrum. Only the 160-isotope of y ions should be retained
in the subtracted spectrum. The limited mass resolution achievable with a triple
quadrupole instrument when used in a high transmission mode limits the quality
of the filter. We interpret spectra acquired with the differential scanning technique by using the overlaid original data. The subtracted spectrum guides the
attention more rapidly to putative y ions. Y ion only spectra are much simpler to
interpret than complete tandem MS spectra. They can in principle be generated
by an orifice fragmentation process followed by a precursor ion scan for the arginine or lysine yl ion (LehmannWD 1998). However, the double fragmentation
process limits the sensitivity of this analysis and it is not possible to work on peptide mixtures.
The differential scanning technique relies on the precise control of the ions
transmitted into the collision zone. Therefore, it is mandatory to have the first
quadrupole Ql correctly calibrated and to adjust the Ql resolution such that the
window of transmission covers a range of approximately 3 Da on the m/z scale.
The 16 0 isotope of the peptides should be correctly assigned in the Ql spectrum.
We always acquire a well resolved Ql spectrum of the peptide mixture before the
tandem MS analysis begins.
It is more difficult to identify y ions with the 33 % 18 0 labeling technique using
differential scanning than when the peptides are methylated. But since the oxygen labelling method is so much easier to use the majority of proteins sequenced
de novo in our laboratory on the triple quadrupole machine are analyzed in this
way.
4.2
Differential Scanning Based de Novo Protein Sequencing
with a Quadrupole-Time of Flight Mass Spectrometer
The advantage of the differential scanning technique is clearly visible for spectra
generated with a quadrupole-time of flight instrument. The high resolution of
the spectra allows to use 50 % 18 0 labeling. The instrument parameters can be
adjusted by observing the isotopes transmitted into the collision cell with a low
collision energy setting before the tandem MS investigation starts.
Subtraction of the 18 0 tandem mass spectrum from the 16 0/ 18 0 spectrum can
yield a clear y ions only spectrum (see Fig. 5.3). The subtracted spectrum has a
far lower complexity compared to the original one. Because of its simplicity and
the high mass accuracy, there is often only one possibility to assemble larger ions
into a series of amino acids. The filtered spectrum can serve as a source of information to interpret tandem MS spectra automatically. The original spectra contain more fragment ions like a, b and internal fragment ions but the subtracted
spectrum helps in understanding the complete fragmentation pattern due to its
relative simplicity. About 50 % of the peptides investigated so far using this technique could be correctly interpreted automatically from the subtracted spectrum
alone. When interpreting the original data together with the subtracted spectrum