Analysis of Post-translational Modifications by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 94

determination of the site and extent of modification is crucial for
deciphering cellular signaling networks. Crucially, both the type
and location of modification within the tertiary protein structure
may influence its function, with the stoichiometry of modifica-
tion potentially determining the extent of any effect. This chapter
therefore discusses, in brief, the current LC-MS/MS methodolo-
gies that can be used to address these questions. For a more com-
prehensive review, readers are directed to Eyers and Gaskell ( 4 ) .

2. Mass
Spectrometry
in the Analysis
of PTMs

Due to its sensitivity and specificity, mass spectrometry (MS) is an
analytical strategy widely used for the identification and charac-
terization of gene products within biological systems. A variety of
MS instruments can be used for the identification and quantifica-
tion of proteins present in complex biological mixtures. Typically,
this requires the separation of proteins and/or their constituent
peptides, often using some form of chromatography, prior to MS
analysis. Mass analysis of a single purified protein can theoreti-
cally permit the identification of the type of PTM present. How-
ever, more detailed analysis, usually at the peptide level, is usually
required to pinpoint the site(s) of modification. A modified pep-
tide derived from such a sample can be detected readily by virtue
of a mass increment or deficit relative to the unmodified form
of the same peptide. Certain modifications induce a consistent
characteristic mass change ( 4 ) . For example, observation of mass
differences representative of phosphorylation (+80 Da), methyla-
tion (+14 Da), or S -nitrosylation (+29 Da) is a good indicator of
modified peptide species. However, presence of a particular mass
difference does not conclusively permit identification of the site
of modification (for which tandem MS is required), although the
site of modification can sometimes be directly inferred if only a
single potential site is present in the putative peptide sequence.
It should also be noted that observation of a defined mass incre-
ment may be indicative of a number of different modifications.
For example, the mass change that arises as a result of trimethy-
lation (+42.05 Da) and acetylation (+42.01 Da) may be indis-
tinguishable when using mass spectrometers with limited mass
accuracy. Similarly, phosphorylation (+79.9663 Da) and sulfa-
tion (+79.9568 Da) can only be distinguished unambiguously
with instruments capable of high mass accuracy or by using
modification-specific analytical strategies such as affinity enrich-
ment or enzymatic (e.g., phosphatase) treatment.
Characterization of PTM sites by LC-MS/MS is currently
achievable using a number of complementary approaches that
can be broadly separated into four categories: (i) tandem MS