Affinity Targeting Schemes for Biomarker Research - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 209

Ga þ 3 ,Al þ 3 ,orFe þ 3 .Ga þ 3 columns seem to give
the highest recovery but are hard to prepare
due to the poor solubility of Ga þ 3 .Fe þ 3 is the
easiest to prepare and most widely used. The
weakness of IMAC columns is that carboxyl-
rich peptides are selected as well. Binding of
carboxyl-rich peptides can be minimized by
digestion with Glu-C instead of trypsin. Another
strategy to minimize the binding of carboxyl-rich
peptides is to overload the IMAC column,
setting up a competition for IMAC stationary
phase groups. 84 This setup is most easily
achieved with small tandem IMAC columns.
The

Ion Exchange
Tryptic peptides generally have little positive
charge above roughly pH 9 but a

2 charge
below pH 7. When phosphorylated, they have
at least a e 3 charge at pH 9, which allows
them to be captured with a strong anion
exchanger. At pH 2.7, roughly 70% of the phos-
phopeptides will have a net charge of

þ

1 and
show less retention on a strong cation exchanger
relative to other peptides ( Figure 6 ).

þ

Acetylation
Acetylation, ubiquitination, SUMOylation, 86
biotinylation, 87 formylation, and methylation
all occur on lysine residues in proteins. 88 In
fact, they even compete with each other, 89 as is
the case with SUMOylation in regulating nuclear
transport of proteins. Protein acetylation on
lysine is a dynamic process known to play an
important role in regulating transcription. At
present, 388 acetylation sites have been identi-

first column overloads while the second
still has binding capacity. In this scenario,
multiply phosphorylated peptides binding on
the

first column while singly phosphorylated
peptides bind on the second. Another strategy
is to use high concentrations of acetonitrile in
the electrospray mode of ionization. At high
acetonitrile, phosphopeptides ionize better
than acidic peptides. Higher iron purity and
desalting before selection provide increased
enrichment.

fied in 195 proteins derived from HeLa cells
and mouse liver mitochondria. 90 More than
20% of mitochondrial proteins carry lysine ace-
tylation sites, including many longevity regula-
tors and metabolic enzymes. Nonnuclear
proteins with a variety of functions, regulators
of chromatin-based cellular processes, and
a series of other nonhistone proteins were identi-

Titanium Oxide
Titanium oxide binds negatively charged
phosphate groups tightly. Singly phospho-
rylated peptides are recovered easily, whereas
multiply phosphorylated peptides are dif

cult
to recover. Acidic peptides are also bound,
which

fied as well.
N-acetylation of lysine is most easily studied
by selecting proteins that are derivatized with
an antibody. 91 Several thousand acetylation sites
in human cancer patients have been identi

cation. 2,5-
dihydroxybenzoic acid or glycolic acid mini-
mizes this problem and increases recovery.
Other substituted aromatic carboxylic acids
were also capable of speci

is reduced by esteri

ed in
this way. 92 Antibodies can be of two types: those
prepared by using a single N-acetylated protein
immunogen or a pan-acetylated protein
mixture. 93 Antibodies derived from a single
immunogen will potentially enrich the immu-
nogen and acetyllysine peptides derived from
it, exhibiting narrow sequence diversity. Pan-
speci

cally reducing the
binding of nonphosphorylated peptides. Phos-
phoric acid reduced the binding of both phos-
phorylated and nonphosphorylated peptides.
TiO 2 is more selective than Fe þ 3 IMAC. 85 Add-
ing glycolic acid to the loading buffer increases
recovery using ammonium hydroxide as the
eluent. LC-ESI-MS/MS is biased toward mono-
phosphorylated peptides, whereas MALDI-MS
is not.

c antibodies can also be obtained by using
a monoclonal antibody cocktail where mono-
clonal antibodies were expressed against