Mapping of Phosphorylation Sites by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 133

100mMMES(pH6.0),and1Mpolyaminesuchaspolyal-
lylamine or PAMAM dendrimer generation 5.
3. Adjust pH to 5.5, if necessary, with 1 M NaOH.
4. Shake vigorously for 12-18 h at room temperature.
5. Transfer the solution to a Biomax filter device with molec-
ular weight of 10,000 Da.
6. In the following steps, mix the polymer/dendrimer well to
avoid unspecific binding of non-phosphopeptides.
7. Wash three times with 500 μ L of 3 M NaCl and discard

the flow-through.
8. Wash two times with 10% methanol and discard the flow-
through.
9. Wash two times with water and discard the flow-through.
10. Add 10% TFA for 30 min to recover phosphopeptides.
11 Wash the polymer with 10% methanol several times and
collect the flow-through.
12. Dry the collected flow-through under vacuum.
13. Resuspend for LC-MS analysis.

3.4. Identification
ofPhosphopeptides
fromLC-MSData
byDatabase
Searching

There are many workflows possible based on different search
engines like Mascot ( 8 ) or Sequest ( 9 ) and different mass spectro-
metric platforms to identify phosphopeptides, but most have the
following in common. After phosphopeptide enrichment, peptide
fractions are separated prior to MS analysis on a reverse-phase-
based column. Typical characteristics of this setup are packing
material of C18, inner diameter of the column 75-125

μ m, par-

ticle size 2-5 m, and an ACN/water/formic acid solvent sys-

tem. Many large-scale phosphoproteomic projects ( 10 - 12 ) have
acquired their data on Fourier transform-based mass spectrome-
ters such as the LTQ-Orbitrap ( 13 ) .
Both phosphopeptide identification and site determination
are affected by frequent losses of phosphoric acid from the pep-
tide, particularly in ion trap CID fragmentation spectra resulting
in a dominant pseudo-neutral loss peptide ion. Often this results
in low intensity of the peptide fragment ions. To combat this phe-
nomenon, several mass spectrometric methods have been devel-
oped, such as MS 3 scans for neutral loss-containing MS 2 ions and
multi-stage activation to further fragment these same ions. How-
ever, it has been recently shown that with high mass accuracy mass
spectrometers, this is no longer beneficial ( 14 ) .
After acquisition of the mass spectrometric data, the raw data
are converted to an appropriate format for the specific search
engine. It has to be noted here that data filtering can have a

μ