Affinity Targeting Schemes for Biomarker Research - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 207

Displacement occurs slowly, generally produces
wide if not multiple peaks in some cases, and
may not completely displace all glycans. The
advantage of glycan displacement is that it is
mild and less likely to desorb non e speci

studying phosphorylation seem not to give the
same answer. But the reason for that will be
found and resolved. Phosphorylation sites are
being identi

ed in primarily four ways: with anti-
body selection, immobilized metal af cally nity chro-

matography, titanium oxide selection, and
through ion exchange ( Figure 6 ). Roughly 5% of
the vertebrate genome codes for kinases and
phosphatases. 79 Phosphorylation occurs most
frequently on serine (

bound species.
Stable isotope coding for quanti

cation can be
achieved in multiple ways, as described in
Figure 2 . Relative quanti cation was used in all

the early glycoprotein studies; more recently,
MRM-based quanti

w 90%) residues, to a lesser

cation was introduced. extent on threonine ( w 10%), and by far the least

cation based on
nonglycopeptides is easiest, quickest, and
involves the fewest steps. Because aberrations
in glycosylation are site speci

Identi cation and quanti on tyrosine ( 0.05%). Although of lowest abun-

dance, tyrosine phosphorylation is often used in
signaling and is reversible, playing an important
role in both normal signal transduction cascades
and aberrant signaling pathways encountered in
malignant disease.

w

c, it is necessary
in many cases to examine peptides from speci c

sites. It is easier to ionize deglycosylated
peptides, but direct examination of glycopep-
tides will have the greatest amount of informa-
tion. Relative standard deviation (RSD) values
for glycopeptide quanti

Antibodies
Antibodies are commercially available for all
three types of phosphorylation, but the anti-pY
antibodies are far superior to those developed
for pS

cation are not available

at the present time.
The number of reported lectin-based glyco-
protein studies is growing exponentially, partic-
ularly in association with cancer. An ovarian
cancer study describes aberrant glycosylation
in the tissue and serum of endometrioid ovarian
cancer patients 72 and another shows similarities
with glycomic mapping of pseudomucinous
human ovarian cyst glycoproteins. 73 Numerous
papers have appeared recently showing putative
glycoprotein markers in breast cancer based on
lectin af

and pT. 80 Biotinylated antibodies

(Ab B) that target pY are available as well.

They are particularly useful

w

in selecting

pY:Ab B complexes from plasma where there

is a large amount of IgG. 81 It was noted earlier
that one of the problems with antibody selection
is that other proteins are selected that either bind
nonspeci

w

nity matrix or are part
of a complex of which one or more proteins
bear a pY. One of the methods of dealing with
this problem is double af

cally to the af

nity selection. 74,75 Increased b 1-6
branching seems to be an important element in
up to 50% of the primary malignancies. 76 Similar
studies have been reported for

nity selection. Subse-
quent to selection of pY-bearing proteins with
an anti-pY immunosorbent, the af

nity-selected
fraction is trypsin digested, phosphate esters on
pS and pT carrying peptides are b -eliminated
in base, and the pY peptides selected with an
Fe þ 3 loaded IMAC column before identi

colorectal

cancer 77 and pancreatic cancer. 78

cation

Phosphorylation
Thereisgoodnewsandbadnewsrelativeto
the analyses of phosphorylation. The good news
is that more than 1,000 phosphorylation sites
have been found in proteins via af

by RPC-MS/MS. 82

Immobilized Metal Affinity
Chromatography (IMAC)
IMAC columns are widely used in the selec-
tion of phosphorylated peptides 83

nity selection.
The bad news is that the various methods for by loading