This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.An aliquot (2.0 mg) of the sample was lyophilized and then the dried sample was dissolved in ammonium bicarbonate buffer (50 mM, pH 8.4) and followed immediately by reduction with 25 mM dithiothreitol (45 min at 50oC) and carboxyamidomethylation with 90 mM iodoacetamide (45 min at room temperature in the dark) prior to trypsin digestion at 37 C for 20 hours. A second enzyme, peptide N-glycosidase F (New England BioLabs) was added to the tryptic digest and incubated at 37 C for 20 hours to release the N-linked glycans. After enzymatic digestions, the sample was passed through a C18 reversed phase cartridge. The N-linked glycan was eluted with 5% acetic acid and then lyophilized.Neutral and amino sugars were released from 255 g (glycoprotein amount) of the N-linked glycans sample submitted through hydrolysis with 2 N trifluoroacetic acid at 100 C for 4 h. After hydrolysis the sample was lyophilized. The dried sample was then resuspended in 150 L water and sonicated on ice. For analysis, 10 L were injected. Blanks (water) were injected and run between the standards and the sample (data not shown) to prevent carry-over. The calibration standards were hydrolyzed in parallel with the sample, and then injected at three levels (0.5, 1.0 and 2.0 nmol). The quantitation was based on a linear interpolation from these standards. Sialic acids were released from 150 L of each of the samples submitted by mild acid hydrolysis using 2M acetic acid at 80 C for 3 h. The sample was dried as above, and resuspended in 150 L water. For analysis, 10 L were injected. Blanks (water) were injected and run between the standards and the sample (data not shown) to prevent carry-over. The calibration standards were hydrolyzed in parallel with your sample, and then injected at three levels (0.5, 1.0 and 2.0 nmol). The quantitation was based on a linear interpolation from these standards.
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