68
M. WILM eta!'
ive Biosystems, Cambridge, USA) assembled in pulled glass capillaries (GC 120,
Clark Electromedical Instruments, Pangbourne, England) (Wilm M and Mann M
1996). The peptide mixture was eluted directly into a nano electrospray capillary
using two times 0.5 [11 of(60% methanol, 5% formic acid, 35% water). The gold
covered nano electrospray capillary was mounted in the nano electrospray ion
source for tandem mass spectrometric analysis.
3.2
Mass Spectrometry
Pep tides were fragmented on a triple quadrupole machine (PE-Sciex, API III,
Ontario, Canada) and two different quadrupole time of flight machines (QqTOFprototype of PE-Sciex and Q-TOF from Micromass, Manchester, UK).
On the triple quadrupole machine spectra were acquired with 0.2 Da stepwidth. The triple quadrupole instrument was controlled in a semi-automated way
using AppleScript™ macros. The fragmentation energy was chosen automatically
depending in a linear way on the m/z value of the precursor and the range of the
fragment spectrum acquired-one setting for the region below and a second, lower
collision energy setting for the region above the precursor m/z value. The width
of the Q1 selection window for the precursor was reduced to transmit less chemical noise ions when acquiring the low m/z region of the fragment spectrum.
In order to acquire the fragment spectrum of the 18 0 isotope the selection in
Q1 was increased by 1 unit on the m/z scale from the m/z value of the first isotope
of the peptide.
In contrast to the acquisitions on triple quadrupole instruments, the instrument parameters were the same for the complete spectrum on the quadrupole
time of flight machines. The 16 0/ 18 0 isotope spectra and the 18 0 isotope spectra
were acquired with identical adjustments. In general the transmission of the
selected isotopes was controlled with low collision energy settings to exclude reliably the 16 0 containing peptide ions before the acquisition of the tandem MS
spectrum.
3.3
Data Processing
Data of the tandem mass spectra were processed using the program packages
IGOR Pro (Wavemetrics, Lake Oswego, USA) and Bio-MultiView (PE-Sciex).
Before processing, the spectra acquired with the quadrupole time of flight
machines were baseline subtracted, those obtained with the triple quadrupole
mass spectrometer smoothed with Bio-MultiView. The data of the two tandem
mass spectra of the different isotopes of a peptide were set to parallel m/z values
in steps of 0.02 Da (QqTOF data) and 0.05 Da (Q- TOF data) using the program
IGOR Pro. For triple quadrupole data the stepwidth was 0.1 Da. The quadrupole
time of flight spectra of different isotopes of the same peptide were scaled to each
other in windows of20 Da and subtracted. For triple quadrupole spectra the scaling window was 10 Da wide. Only positive data values were exported to generate
the subtracted spectrum. Subtracted spectra were noise filtered in Bio-MultiView
M. WILM eta!'
ive Biosystems, Cambridge, USA) assembled in pulled glass capillaries (GC 120,
Clark Electromedical Instruments, Pangbourne, England) (Wilm M and Mann M
1996). The peptide mixture was eluted directly into a nano electrospray capillary
using two times 0.5 [11 of(60% methanol, 5% formic acid, 35% water). The gold
covered nano electrospray capillary was mounted in the nano electrospray ion
source for tandem mass spectrometric analysis.
3.2
Mass Spectrometry
Pep tides were fragmented on a triple quadrupole machine (PE-Sciex, API III,
Ontario, Canada) and two different quadrupole time of flight machines (QqTOFprototype of PE-Sciex and Q-TOF from Micromass, Manchester, UK).
On the triple quadrupole machine spectra were acquired with 0.2 Da stepwidth. The triple quadrupole instrument was controlled in a semi-automated way
using AppleScript™ macros. The fragmentation energy was chosen automatically
depending in a linear way on the m/z value of the precursor and the range of the
fragment spectrum acquired-one setting for the region below and a second, lower
collision energy setting for the region above the precursor m/z value. The width
of the Q1 selection window for the precursor was reduced to transmit less chemical noise ions when acquiring the low m/z region of the fragment spectrum.
In order to acquire the fragment spectrum of the 18 0 isotope the selection in
Q1 was increased by 1 unit on the m/z scale from the m/z value of the first isotope
of the peptide.
In contrast to the acquisitions on triple quadrupole instruments, the instrument parameters were the same for the complete spectrum on the quadrupole
time of flight machines. The 16 0/ 18 0 isotope spectra and the 18 0 isotope spectra
were acquired with identical adjustments. In general the transmission of the
selected isotopes was controlled with low collision energy settings to exclude reliably the 16 0 containing peptide ions before the acquisition of the tandem MS
spectrum.
3.3
Data Processing
Data of the tandem mass spectra were processed using the program packages
IGOR Pro (Wavemetrics, Lake Oswego, USA) and Bio-MultiView (PE-Sciex).
Before processing, the spectra acquired with the quadrupole time of flight
machines were baseline subtracted, those obtained with the triple quadrupole
mass spectrometer smoothed with Bio-MultiView. The data of the two tandem
mass spectra of the different isotopes of a peptide were set to parallel m/z values
in steps of 0.02 Da (QqTOF data) and 0.05 Da (Q- TOF data) using the program
IGOR Pro. For triple quadrupole data the stepwidth was 0.1 Da. The quadrupole
time of flight spectra of different isotopes of the same peptide were scaled to each
other in windows of20 Da and subtracted. For triple quadrupole spectra the scaling window was 10 Da wide. Only positive data values were exported to generate
the subtracted spectrum. Subtracted spectra were noise filtered in Bio-MultiView