Methods to improve detection of glycosylamines
A glucosylamine, amine-reactive technology, applied to the composition of analytes, in the field of analyzing the labeled glucosylamine, which can solve the problems of tediousness and unsuitability for automation
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Embodiment 1
[0082] This example sets forth abbreviations for some of the reagents used in the example workflow of the labeling procedure using example carbohydrates in some examples below.
[0083] "SDS": Sodium Dodecyl Sulfate
[0084] "Tris": Tris(hydroxymethyl)aminomethane
[0085] "Peptide N-Glycosidase F Mixture": Peptide N-Glycosidase F (about 1 mg / ml) and 750 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH 8.0 buffer 1 :1 mixture.
Embodiment 2
[0087] This example sets forth the N-glycan InstantPC for use with exemplary amide supports TM An example workflow for markup.
[0088] N-glycan release and preparation
[0089] Four samples of 10 μl of the 4 mg / ml exemplary glycoprotein etanercept were added to the wells on the PCR plate. 2 μl of SDS was added to each well and the PCR plate was incubated at 90° C. for 3 minutes to denature the glycoproteins. After cooling the samples to below 50°C, 2 μl of Peptide N-Glycosidase F mix was added to each well to cause enzymatic digestion of glycoproteins and release of glycans from glycoproteins as glucosylamines. The PCR plate was then incubated at 50°C for 5 min. Then 10 μl of 1.5M Tris pH 8 buffer was added to each of the two samples to achieve a final concentration of 750 mM Tris, while 10 μl of H 2 O was added to each of the other two samples to keep the volumes of the four samples the same. The volume of each sample was approximately 26 μl.
[0090] Labeling and subs...
Embodiment 3
[0096] This example reports the results of the study set forth in Example 2.
[0097] The results are graphically depicted in Figure 2. The two bars on the left of the graph show the fluorescence of the labeled sample in solution. The control (sample with only water added to the deglycosylation mixture) showed a peak area of over 35 million units, while the sample containing Tris buffer showed a peak area of about 2 million, reflecting that when Tris buffer was present in the sample or could Another source of amines that react with amine-reactive dyes is the difficulty in detecting glucosylamine. The two columns on the right side of the graph show the fluorescence of samples that were placed in 85% organic solvent / 15% aqueous solution, immobilized on a hydrophilic solid support, labeled on the support, eluted and analyzed. The second bar from the right shows the results for the control (water only added to the deglycosylation mixture) and shows a peak area of nearly 33...
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