Analysis of Post-translational Modifications by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 98

observation of improved sequence coverage when highly charged,
generally larger peptides are subjected to ETD has encouraged
proteolytic digestion with enzymes other than trypsin. Lys-C,
which cleaves C-terminal to lysine resides, typically generates
larger peptides than trypsin (which cleaves C-terminal to both
lysine and arginine). The application of Lys-C digestion in con-
junction with ETD has enabled better fragmentation, higher
sequence coverage, and therefore increased PTM site discovery
when compared to procedures using tryptic peptides ( 22 , 23 ) .
Although ETD and ECD have proved extremely useful at over-
coming some of the PTM-associated neutral loss issues observed
during CID, these alternative fragmentation mechanisms do not
completely overcome these problems. Sulfopeptides are more sus-
ceptible to neutral loss than are phosphopeptides of identical
amino acid sequence, both during ECD and the marginally “gen-
tler” process of ETD ( 24 ) . However, it appears that the amount
of neutral sulfate loss can be reduced (and therefore site identifi-
cation improved) by fragmenting the peptide-alkali metal adduct
as opposed to the protonated peptide ions ( 24 ) .

3. Selective
Enrichment

The highly dynamic nature of many PTMs often means that the
stoichiometry of modification is relatively low, further compli-
cating their characterization. Additionally, the efficiency of ion-
ization and/or detection of modified peptide species (notably
phosphopeptides) ( 25 ) , when compared to their non-modified
counterparts, can be compromised. Strategies have therefore
been developed that target the explicit identification of pep-
tides or proteins carrying specific post-translational modifications
( Fig. 5.3 ) . Selective enrichment techniques fall within three gen-
eral categories: (i) incorporation of a tagged modification for
selective enrichment; (ii) affinity purification using specific biolog-
ical agents, and (iii) affinity enrichment based on chemical func-
tionality of the modification.

3.1.Tagging

Large-scale analysis of sites of ubiquitination has been achieved
using a strain of Saccharomyces cerevisiae expressing only a His 6 -
tagged form of ubiquitin. Ubiquitin-conjugated proteins were
then affinity purified using nickel chromatography, prior to pro-
teolysis with trypsin, SCX peptide separation, and LC-MS/MS.
This approach was successful, identifying over 1,000 ubiquitin-
modified proteins and characterizing 110 sites of ubiquitination
in a single study ( 26 ) . Whilst approaches such as these that rely
on using specifically designed modified strains of an organism of
interest undoubtedly provide large quantities of data for further