Biology Reference
In-Depth Information
OrbiTrap mass analyzer were isolated with an isolation width of
2 Th in the linear trap quadrupole (LTQ) mass analyzer and
fragmented applying collision induced dissociation (CID). The
fragment ions were then recorded in low resolution MS/MS
spectra. Electron transfer dissociation (ETD) fragmentation is
recommended when available because it leaves the phosphate-
ester bond intact thereby preserving the phosphate moiety in
peptide primary structure and generates extensive fragmentation
of the peptide backbone for MS/MS spectra rich in sequence
information [
4
]. Automatic gain control (AGC) for the Orbitrap
was set to 5e+05. The Maximum injection time (max IT) was set
to 500 ms, and injection wave forms were enabled. Full scan
mass spectra were internally calibrated on the fl y using lock mass
for an average mass error of less than 1 ppm. Exclusion duration
for data-dependent selection of MS/MS precursor ions was set
to 30 s and exclusion width was ±10 ppm. Exclusion duration of
30 s may seem low but we thought it suitable to increase the
number of MS/MS spectra acquired for low abundant phospho-
peptides. Charge state screening was enabled and single-charged
ions were excluded from MS/MS acquisition. Multistage activa-
tion/pseudoMS3 was enabled with a neutral loss mass list of
293.91, 195.94, 97.97, 48.999, 32.66, and 24.49 Da to gener-
ate concatenated MS/MS spectra containing peptide backbone
fragments when initial CID resulted in release of the phosphate
moiety. The AGC for MS/MS acquisition in the LTQ was set to
3e+04; the max IT was set to 100 ms. Three microscans were
acquired per MS/MS spectrum.
8. Control all LC-MS system and method parameters, the sample
list, the vial position in the auto sampler and start the analysis.
1. Load raw fi les. MaxQuant accepts .raw fi les from Thermo
Scientifi c mass spectrometers. MaxQuant version 1.3.0.5
accepts mzXml fi les so it is compatible with other instrument
vendors. Convert the mass spectrometer's output fi les to
mzXml format if necessary. There are several tools available on
line such as RAW Xtractor as part of the Census package from
the Scripps Institute, DTA Supercharge integrated into
MSQuant or MM File Conversion as part of the MassMatrix
database search engine for this purpose. Some diffi culties con-
fi guring these software to individual systems may arise.
2. Generate an experimental design template and then load it
into MaxQuant under the tab “Identifi cation & quantifi ca-
tion.” We recommend declaring each LC-MS analysis includ-
ing replicate analyses as an individual experiment. Grouping
LC-MS analyses will produce a summed intensity over all
grouped analyses for each identifi ed phosphopeptide and will
preclude estimation of variability between individual analyses.
3.6 Phosphopepide
Identifi cation,
Quantifi cation, and
Phosphosite Mapping
( See Note 2 )
