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: Aliquot of each sample (100?g) were used for N-linked profiling. Samples were denatured first and digested with trypsin, followed by glycopeptides enrichment by C18 sep-pak. N-glycans were then released from the glycopeptides enzymatically by PNGase F. Released N-glycans were separated from residual contents by C18 sep-pak and permethylated prior to mass spec analysis. Detailed procedures used for your sample analysis are shown in detail below. N-linked glycan preparation An aliquot (100?g) of each sample was dried and reconstituted with 0.1 M Tris-HCl buffer, pH 8.2 containing 0.01 M CaCl2. The sample then was denatured by heating for 5 minutes at 100?C. After cooling, the sample was digested with the trypsin (37oC, overnight). The sample was then heated at 100? C for 5 min to inactivate trypsin. The sample was then passed through a C18 sep-pak cartridge and washed with 5% acetic acid to remove contaminants (salts, free sugar, etc.). Peptides and glycopeptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol and dried in a speed vacuum concentrator. The dried samples were combined and incubated with PNGase F at 37?C overnight to release N-glycans. After digestion, the sample was passed through a C18 sep-pak cartridge and the released N-glycans was eluted with 5% acetic acid and dried by lyophilization, and then permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) and profiled by mass spectrometry. Mass spectrometry MALDI/TOF-MS was performed in the reflector positive ion mode using ?-dihyroxybenzoic acid (DHBA, 20mg/mL solution in 50%methanol:water) as a matrix. The spectrum was obtained by using a Microflex LRF (Bruker).
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