Approaches and Applications of Quantitative LC-MS for Proteomics and Activitomics - LC-MS/MS in Proteomics: Methods and Applications - page 5

signalling ( 9 ) . It was found that >2,000 phosphorylation sites are
regulated as a result of activating the EGF receptor in HeLa cells,
which indicates that kinase pathways downstream of growth fac-
tors are more complex than previously thought. These studies
used a combination of SCX and TiO 2 to enrich phosphopeptides
that were later identified and quantified by LC-MS/MS. Related
strategies involve the use of IMAC instead of TiO 2 as the enrich-
ment step ( 12 , 13 ) or the use of anti-phosphotyrosine antibodies
to enrich phosphotyrosine-containing peptides ( 10 , 11 ) . Using
these protocols (detailed in Chapters 6 and 7 ), it is now possible
to 'routinely' identify and quantify thousands of phosphopeptides
per experiment.
A question that need to be addressed is what is the value
of these analyses ( 14 ) ? Indeed, these authors believe that the
significance of phosphoprotein quantification is sometimes not
fully understood. A misconception often found in the literature
is that phosphorylation activates the enzymatic activity of pro-
teins. Although this is the case for many enzymes, including pro-
tein kinases such as PKB/Akt and Erk1/2, it is not the case for
many others such as GSK3

, which is inactivated as a result of
being phosphorylated by PKB/Akt. It has also been proposed
that much of the phosphorylation occurring in eukaryotes is
non-functional ( 14 ) , with functional events occurring in a back-
ground of phosphorylation 'noise' for which there is no selec-
tion against. An example of apparent non-functional phosphory-
lation is that occurring on PKB/Akt, which although phosphory-
lated at Ser124, Ser126, Ser129, Thr308, Ser473 and Tyr474 (of
the human AKT1 isoform), only the phosphorylations at Thr308
and Ser473 are known to modulate the enzymatic activity of
PKB/Akt. The other phosphorylation sites do not seem to alter
any of the properties of this kinase.
The hypothesis of the existence of non-functional phospho-
rylation is difficult to prove or disprove, because phosphoryla-
tion with no apparent function under determined conditions may
have functions under other conditions. It should also be noted
that activation of enzymatic activity is only one of the proper-
ties that phosphorylation may attribute to a protein. Indeed, not
all proteins are enzymes and phosphorylation can also affect pro-
tein localization and their ability to interact with other molecules
in cells, which may be as important in controlling the biological
properties of proteins as the modulation of enzymatic activity.
Regardless of whether or not non-functional phosphoryla-
tion exists, in our view, the value of phosphoproteomics lies in
its potential ability to serve as a comprehensive read-out of kinase
activity in cells. There exist 518 protein kinase genes in cells, and
the deregulation of these enzymes is a common occurrence of
many diseases including cancer, where kinases have been found to
be frequently mutated ( 15 ) . But because enzymatic activity can be

β