Analysis of Post-translational Modifications by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 93

The most commonly analyzed PTMs are covalent modifica-
tions, largely because of the close association between their dys-
regulation and a variety of disease states, and due to the bio-
logically important changes in protein function that can occur
as a result of their addition or removal. Covalent PTMs encom-
pass both chemical moieties such as phosphate and carbohydrates,
and functional polypeptides such as ubiquitin and SUMO. The
reversible addition of a chemical moiety such as phosphate, cat-
alyzed by specific protein kinases, plays an essential role in regu-
lating signaling in both prokaryotic and eukaryotic cells. Incor-
poration of one or more phosphate groups on specific amino
acid side chains within a protein, with serine, threonine, tyrosine,
and histidine being the most commonly studied, often induces
significant protein conformational change and consequently pro-
found effects on protein activity and protein-protein interactions
( 2 - 4 ) . Protein glycosylation is significantly more complex, with
two main mechanisms for covalent binding of the glycan to the
polypeptide: N -glycosylation, where the glycan is attached to
an Asn residue within a tripeptide consensus sequence (Asn-X-
Ser/Thr) (where X represents any amino acid except Pro) and
O -glycosylation, in which the glycan is attached to a Ser or a Thr
residue ( 5 - 7 ) . Protein glycosylation is functionally important for
modulating inter- and intracellular protein activities, cell adhe-
sion, coordination of immune functions, and is critical for mitosis
and cell division.
The functional significance of protein ubiquitination is depen-
dent on the number of ubiquitin monomers attached at a single
site, as well as the site of ubiquitin chain linkage. Ubiquitin is a
76-amino acid (

8 kDa) protein that is covalently attached via its
C-terminal carboxyl group to the

∼

-amino groups of lysine
residues on target proteins ( 8 ) . Poly-ubiquitination via Lys48
of ubiquitin plays an essential role in proteasome-mediated
induced protein degradation, while mono-ubiquitination regu-
lates enzyme activity, protein-protein interactions, and protein
trafficking ( 8 , 9 ) .
Protein function and/or stability can also be altered by spe-
cific structural changes to the polypeptide chain; disulfide bonds
between cysteine residues separated in primary sequence often
help to stabilize tertiary structure. Additionally, amino acid side
chains can be altered. For example, the deamidation of asparagine
generates aspartate, which fundamentally changes the amino acid
composition and charge of the polypeptide post-translationally.
Many of the PTM events that occur on a single protein are known
to be synergistic; phosphorylation can promote protein ubiquiti-
nation, thereby coupling an extracellular stimuli-initiated signal
transduction event with subsequent protein degradation ( 8 ) .
Over 300 PTMs have been reported in the literature ( 1 )
and since they regulate all aspects of cellular homeostasis,

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