Chemistry Reference
In-Depth Information
removed under acidic conditions. Weak acid lysis at pH 2.0 and 90 ° C for 60 min
is used to release the sialic acid. Weaker acidic conditions in solution or on ion-
exchange resins can result in the loss of sialic acids. Sialic acid residues are also
displaced naturally from the glycan at a low pH by its own acidity in concentrated
sialyl - glycan solutions. Purifi cation methods are selected on the basis of the par-
ticular character of the glycoprotein and the purpose of the study.
Carbohydrates do not exhibit ultraviolet absorption, which is another major
analytical problem. Glycans are detected by refractive index, pulsed amperometric
detection and UV detector at 210 nm using HPLC. However, these methods also
detect other biomaterials so that they require tight restrictions when used for
analysis, and are often not suffi ciently sensitive. Various derivatization methods
have been developed for the detection of oligosaccharides [7] and most of them
are based on the reaction of the reducing ends with aldehyde (Figure 5.3 ). Pyridyl-
amination, developed by S. Hase in 1978, was the fi rst labeling technique to use
reductive amination of the reducing end, and it is still widely used in HPLC and
MS. The labeling of glycans not only increases the sensitivity of HPLC through
its fl uorescence, but also enhances the separation obtained using reversed- phase
columns. Improved separation of oligosaccharides including isomers through
labeling is of great advantage, as the sensitivity of MS is dependent on the purity
of the sample glycans. Derivatization for sialic acid or permethylation is also used,
not only to resolve problems of sensitivity, but also to increase the stability of sialic
acids during MS analysis [8] .
5.4
Detailed Structural Analysis Using HPLC
A widely applied approach to analyze oligosaccharide structure uses the elution
positions on two- or three-mode columns. Two-dimensional mapping is a strategy
used for neutral
N
- glycans employing pyridylamino (PA) labeling and HPLC on
two types of column, octadecylsilica (ODS) and amide [9]. To increase reproduc-
ibility, elution position is then converted to glucose units, which are relative
elution positions compared to a glucose oligomer. Several PA-glycans are com-
mercially available, and they help to increase reliability of the elution positions and
subsequent enzymatic treatment (for further application of PA-glycans, please see
Chapter 14.3). To analyze sialyl-glycans and clarify the number of sialic acid resi-
dues, a third dimension using a diethylaminoethyl column is applied [10] . Isomers
are separated on the ODS column and interactions on the amide column are cor-
related with the size of the glycans. The
N
-glycan structure is indicated by the
elution position on the two or three columns when compared with elution posi-
tions of about 500 known oligosaccharides (http://www.glycoanalysis.info/ENG/
index.html). We can therefore derive the structure of the sample
N
- glycan corre-
sponding to these elution positions using a database. The fl uorescence intensity
of the PA-
N
-glycan does not depend on the
N
- glycan structure and quantitative
analysis is done using UV absorption. The amount and molar ratio of the
N
- glycan
can be subsequently calculated.
