Biology Reference
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palmitoylation. Proteins can also be proteolytically modified after translation,
although this is often considered to be functionally separate from chemical
addition of groups to the protein backbone or side chains. The chemical PTMs
can either be rapidly reversible (e.g., phosphorylation) or long lasting
(e.g., ubiquitination). This varied nature of protein PTMs underlies their
importance in cellular signaling pathways, as thesemodifications allow the gen-
eration of multiple diverse GPCR ligand controllable alterations in protein
signaling networks. Detection of the diverse, subtle, and dynamic protein
PTMchanges that occur in GPCR signaling networks poses a significant chal-
lenge to even the most advanced mass spectrometers. As we have seen in the
previous section, even the reliable mass spectrometric detection of the same
protein between samples is problematical, let alone its specific diverse intact
PTMstate. AsMS techniques assess peptide identities via a combinationof their
mass to charge ratio (
m/z
), the variety of masses or charges of a single peptide
amino acid sequence can be hugely variant with different PTM events. For a
single analyzed peptide, phosphorylated versions may possess the same peptide
backbone but distinct ionization profiles (neutral mass loss), while
palmitoylated peptides may be 238 Da heavier than the same sequence non-
modified peptide.
117
As the range of protein PTMs is extremely diverse, this
has resulted in the general inability to create a single unified workflow that
allows the simultaneous identificationofmultiplePTMtypes ina complex sam-
ple. Therefore, multiple technical approaches need to be applied to extract as
muchPTMinformation fromany startingmaterial. Employingmultiple simul-
taneous approaches reduces MS workflow speed and also may require hugely
different amounts of input sample, depending on the relative abundance in the
sample of each PTM under investigation. Affinity-based enrichments,
immunopurification, two-dimensional differential in-gel electrophoresis,
and metal affinity chromatography are commonly used strategies for the
purification of proteins containing specific PTMs. Immobilized metal affinity
chromatography (IMAC) purification is a common chemical affinity strategy
for the enrichment of phosphoproteins, whereby immobilized metal ions
selectively bind to the phosphorylated peptides.
16,118,119
The isolation of
phosphopeptide species perhaps represents the lead technological platform at
the present time for proteomic PTM analysis of GPCR signaling networks.
Protein phosphorylation is one of the most important and dynamic PTM
required for the regulation of most intermediary metabolism signal trans-
duction cascades.
120
An enormous range of protein kinases and phosphatases,
many of which are associatedwith
b
-arrestin-dependent cascades, controls the
phosphorylation status of proteins that result in changes of protein-protein
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