Biology Reference
In-Depth Information
A: PTM enrichment
enrichment of
PTM-bearing
peptides
digestion
O
P
lectin
mAB
-
O
TiO
2
antibody-based
p-Tyr, ac-Lys, diGly-Lys
ionic interaction
phospho-peptides
affinity ligand
glyco-peptides
B: PTM identification and localization
C: PTM site occupancy
altered
PTM level
b
1
b
2
b
3
b
4
b
5
b
6
b
7
b
8
b
9
peptide mass
increased by
mass of the PTM
T
H
E
P
E
P
T
I
D
E
y
9
y
8
y
7
y
6
y
5
y
4
y
3
y
2
y
1
stimulus
Δ
m
Δ
m
Δ
m
mass of some
fragments
shifted by Δm
fraction of the protein
modified at a given site
modified
peptide
occupancy · ratio(
/
)
T
H E P E P
T
I
D
E
+ (1 - occupancy) · ratio(
/
)
localization
score
= ratio(
/
)
unmodified
peptide
T
H E P E P T I
D
E
protein
FIGURE 1.5
Analysis of post-translational modifications by MS. A: PTM enrichment: Substoichiometric PTM-bearing proteins or peptides are
enriched using various strategies, including PTM-directed antibodies, metal ion complexation and affinity ligands. B: PTM identification and locali-
zation: MS directly measures the presence of a PTM by a defined corresponding shift in mass of the peptide and PTM location within the peptide is
obtained by the MS/MS pattern with single amino acid resolution. C: PTM site occupancy represents the fraction of a protein that is modified at a given
PTM site. Site occupancies can be calculated if one can quantify changing amounts of a modified peptide, the corresponding unmodified peptide and the
entire protein in a perturbed system.
datasets. In principle, this can be addressed by targeted
methods in which the mass spectrometer is directed to
acquire data for a particular set of modified peptides
[162]
.
Quantification of PTM sites is achieved in the same
ways as for non-modified peptides. However, this becomes
more complicated when a single peptide has multiple
modification sites. By employing quantification at different
time points, kinetic maps of PTM-site dynamics in
response to various perturbations can be obtained
[67,163,164]
. PTM-level information can be combined
with information on protein levels, as we have recently
shown in a combined phosphoproteomic and proteomic
analysis of the cell cycle
[67]
. However, when measuring
early signaling changes, for example downstream of
receptor tyrosine kinase activation, one usually assumes
that proteomic changes will be minimal and that observed
quantitative changes in phosphopeptides can directly be
attributed to changes at the modification site level.
Furthermore, it may be desirable in a systems biology
context to quantify not only the relative change of a modi-
fication site but also the fraction of the protein that is
modified at this site (
Figure 1.5
C). First reports of the large-
scale determination of phosphorylation stoichiometry have
recently appeared
[165,166]
. Thus, with ever improving
technology, proteomics can now deliver key parameters
on PTMs that are important for cellular modeling, such
