|
Structural and qualitative analyses
|
TLC |
Resolves oligoSia from polySia chains. Applied to study the DP of oligoSia composed of different Sia isomers Easy adaptability and inexpensive.
|
|
(116) |
| MALDI-TOF MS |
Determines exact mass composition and DP of polySia. Preferential detection of unmodified peptides and partial or complete suppression of glycopeptides. Low quantities (ng) of polysialylated proteins can be analyzed. Discerns between α2,9 linked polySia from α2,8 linked polySia. PolySia of DP up to 100, and 40 Sia units have been successfully detected.
|
Poor tolerance to sample impurities. Remotion of peptides and enrichment of glycopeptides is needed. The mass accuracy and the resolution of the signals are reduced in the linear mode and did not allow an identification of incompletely lactonized species.
|
(117) |
| ESI-MS |
Determines linkages and structure. Low quantities from 10 to 20 pmol of the compound with Sia moieties can be analyzed. Sia dimers, trimers, and tetramers can be detected with higher efficiency.
|
|
(118, 119) |
|
Structural and quantitative analyses
|
HPAEC-PAD |
Allows detection of all non-volatile and most semi-volatile analytes. It is not necessary to derivatize samples. PolySia can be quantified by coupling HPAEC with a detector based on amperometry, fluorescence, UV absorbance, or mass spectrometry.
|
To detect DP 50 is necessary 10 μg of purified polySia samples. Epimerization and degradation of carbohydrates. Unstable baseline, loss of sensitivity, and requirement of a dedicated base compatible HPLC.
|
(120, 121) |
| HPAEC-FD |
Widely employed method. High sensitivity. Can detect polySia with DP > 90 Amount of 200 ng derivatized colominic acid have been analyzed. Can also detect polySia from tissues with DP ranging from 18 to 60
|
|
(122) |
| HPAEC-UV |
|
Poor selectivity using short UV wavelength (210 nm) by increasing background. High sample purity is necessary. Among 10 μg purified polySia for analysis
|
(123, 124) |
| HPAEC-CAD |
CAD separates polySia of higher degree of polymerization >90 colominic acid units. Can detect DP among 65 and 130 Sia residues. Does not require derivatization. Less time than other methods. Less specific and less sensitive than HPAED-FD. Lower amount (250 ng)
|
Large amount of polySia polymer is necessary. Restricted to volatile buffers Decreased resolution by increased salt. High standards of sample purity, compared to fluorometric detection.
|
(125–127) |
|
Quantitative analysis
|
ELISA |
Reproducible and reliable method. High specificity and sensitivity. Can analyze very small samples. Rapid and accurate for quantitation of total polysialylated proteins.
|
|
(128) |
| Flow cytometry |
Can detect polySia on the surface of intact cells. The anti-PolySia antibodies and Endo N-GFP fusion proteins can be used in flow cytometry allowing the analysis of the number of polySia positive cells. It is selective and sensible.
|
To corroborate PolySia antibody specificity, sometimes it is necessary to use the Endo N enzyme. Specificity is associated with antibodies and controls must be used. The exact DP is not possible to determine.
|
(129, 130) |
| Fluorometric C7/C9 |
Highly sensitive and selective analysis of internal Sia residues of oligo- and polySia. Internal Sia residues that remain unaffected can be analyzed by HPLC-FD after fluorescence derivatization. Detection of 1-ng amounts of internal Sia residues of oligo- and polySia molecules.
|
False positive quantitative results for internal Sia residues of polySia. The method allows oxidation of α2,9 linked polySia.
|
(131, 132) |
|
Semiquantitative or qualitative analysis
|
Western blotting |
Expression levels of polysialylated-proteins. Associated chemiluminescent and/or fluorescent signals. High specificity. Detects polySia residues with 735 antibody and 12E3 antibody recognizes oligo/polySia.
|
|
(58) |
