Affinity Targeting Schemes for Biomarker Research - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 206

TABLE 1 Selectivity of commercially available lectins (cont'd)

Lectin name Group specificity Glycan(s) selected

Maackia amurensis II (MAA II) Neu5Ac-speci c Neu5Ac a 2-3Gal b 1-4Glc/

GlcNAc

Sambucus nigra (SNA) Neu5Ac-speci c Neu5Ac a 2-6 Gal/GalNAc

Cicer arietinum (CPA) Lectins with complex speci cities Complex structure, binding

inhibited by lgM, fetuin

Euonymus europaeus (EEA) Lectins with complex speci cities Gala 1-3(Fuca1-2)Galb1-3/

4GlcNAc

Phaseolus vulgaris erythroagglut.
(PHA-E)

Lectins with complex speci cities N-linked bi-antennary

Phaseolus vulgaris leukoagglut.
(PHA-L)

Lectins with complex speci cities N-linked tri/tetra-antennary

and can be applied in serial fashion for identifi- -

cation of multiple glycans within the same
protein. 51 It is in this arena that lectins are
coming to play an increasingly important role
in glycoproteomics and identi

success of these early af nity selection methods

in identifying glycoprotein biomarkers and
assessing treatment ef

cacy through changes in
glycoprotein biomarker patterns came from the
use of immobilized lectin selection of speci cation of glyco-

sylated disease marker proteins.
Based on the preproteomics era history of lec-
tins in the isolation of glycoproteins, they were
obvious candidates for targeted glycoproteo-
mics. Using well established lectin selection
strategies, most of the basic LC-MS/MS methods
used in glycoproteomics today arose in the
2001 e 2003 time frame, 4,68,69 similar and simulta-
neous with multidimensional protein identi

c
glycan conjugates associated with cancer, stable
isotope coding of control and experimental
samples for quanti

cation, enzymatic removal
of glycans with incorporation of H 18 O into
aspartic acid to identify N-linked glycosylation
sites, and use of mass spectrometry to identify
peptides. 70 Moreover, af nity selection can still

be coupled with MudPIT. 71
Variables in lectin af

ca-
tion technology (MudPIT) 70 but for targeted
proteomics instead of the shotgun strategy. The
difference between the targeted multidimen-
sional protein identi

nity chromatography e
mass spectrometry (LAC-MS) detection, identifi- -

cation, and quanti cation methods are (1) the

mode of elution, (2) the isotope coding method
used in quanti

cation technologies and
MudPIT is in the selectivity. Af

cation, and (3) whether protein

nity selection
allows structural targeting of the species being
selected in the

identi cation is based on a nonglycopeptide,

a glycopeptide, or a deglycosylated peptide.
Elution of glycopeptides and glycoproteins can
be achieved in either of two ways. One is by
using an acidic mobile phase such as 0.1 M
glycine/2% acetic acid e HCl buffer, pH 2.5. Gly-
coconjugates are quickly eluted in a single sharp
peak with acidic mobile phases that appear to
disrupt lectin structure. The second approach
to elution is use of a sugar or glycan displacer.

rst separation dimension,

whereas the first dimension cation exchange

mode of MudPIT adsorbs all peptides derived
from trypsin digestion of a proteome. The
complexity of tryptic digests sent on to the
RPC column and MS will be 10- to 100-fold
simpler with af nity selection, depending on

the breadth of lectin selectivity. The immediate