Analysis of Post-translational Modifications by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 103

or a binding affinity. However, mass spectrometric signal inten-
sity is dependent not only on the amount of analyte but also on
its chemical properties. A comparison of the signal intensities of a
peptide and its modified counterpart is therefore not an accurate
representation of the proportion of peptide that is modified. Cir-
cumventing the potential issues of changes in ionization efficiency
of differentially modified peptides of the same primary sequence,
relative quantification of stoichiometry between two or more sys-
tems can be achieved using stable isotope labeling. Internal refer-
ence peptides that are chemically identical to the analyte of inter-
est, but can be differentiated by mass due to the incorporation of
heavy-isotope forms of particular elements ( 13 C, 15 N), are there-
fore used for the quantification of different peptide states. Abso-
lute quantification of phosphorylated and ubiquitinated proteins
has been achieved using isotope dilution methods ( 62 - 65 ) . In the
case of phosphorylation stoichiometry determination, phospho-
rylated isotope-labeled surrogate peptides are synthesized, quan-
tified, and added to the sample of interest in known amounts,
permitting calculation of the stoichiometry of modification of the
native peptide based on comparable signal ion intensity. Stable
isotope labeling with amino acids in cell culture (SILAC) permits
calculation of the relative amounts of modified peptides present
between multiple cell populations. This strategy has the advantage
of permitting the combination of samples undergoing analysis at
an early stage in the sample preparation workflow, minimizing
artifactual differences, and is capable of determining the relative
stoichiometry of a large number of modified peptides simultane-
ously ( 66 - 68 ) . However, to specifically assess the stoichiometry
of modified peptides, SILAC is best used in conjunction with one
(or more) of the enrichment or MS-targeted strategies described
previously ( 34 ) .
As an alternative to stable isotope labeling, normalized ion
currents can also be used for the relative quantification of mod-
ified peptides between LC-MS/MS runs. The relative amounts
of modified peptides in two or more samples are then calcu-
lated using variations in the signal ion intensities of peptide
species of interest ( 69 ) , although this strategy is generally not as
precise.
The continued interest of the scientific community in improv-
ing our ability to determine the presence and stoichiometry of
sites of modification ensures that LC-MS/MS-based methodolo-
gies are constantly under development. However, it is only by
characterizing these modified proteins as intact polymers, rather
than as tryptic peptides that the community currently favors,
that it will eventually be possible to assess the true extent of
post-translational modifications and their influence on cellular
function.