This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: The samples were lyophilized and redissolved with 100 mM Tris-HCl and digested with trypsin and chymotrypsin at 37oC overnight. After protease digestion, each of the samples was passed through a C18 column to recover the glycopeptides. The glycopeptides were first treated with a neuraminidase that is specific to cleave 2,3-linked NANA and digestion was undertaken at 37oC for exactly 1 hr. The digests were passed through C18 column thereafter and the 2,3-linked NANA were eluted with 5% acetic acid and lyophilized. The residual glycopeptides were eluted with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid, and 100% isopropanol. The latter eluates were evaporated of isopropanol and eventually lyophilized. The residual glycopeptides were finally treated with a general neuraminidase and incubated at 37oC overnight to cleave 2,6-linked NANA. After the final sialidase digestion, NANA from each digest was eluted with 5% acetic and subsequently lyophilized. The dried 2,3-linked NANA, and 2,6-linked NANA from each sample were dissolved with nanopure H2O, sonicated in ice for 5 min and transferred into injection vials for sialic acids analysis. A mix of sialic acids standards with known number of moles was prepared and diluted serially into four concentrations to establish a calibration equation. The number of moles of each residue in the sample was quantified by linear interpolation from the calibration equation. The sialic acids were analyzed by HPAEC using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector, and an autosampler. The individual sialic acids were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The gradient program used the following mobile phase eluents: 100 mM NaOH, and 1M sodium acetate in 100 mM NaOH. Injections were made every 40 min. All methods were based on protocols described by Hardy and Townsend (Hardy, M. R., and Townsend, R. R., """"""""High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates"""""""", 1994, Methods Enzymol. 230: 208-225).
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