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: Protein rich powder was prepared from each type of kidneys by following the method described in previous report (MH021710Z). O-glycans were released from protein rich powder by b-elimination. The released O-glycans were permethylated and profiled by mass spectrometry. The detailed procedures used for your sample analysis are shown below. Preparation of protein rich powder from kidneys Kidneys were homogenized and de-lipidated followed by the method of Aoki.et.al (2007). Briefly, kidneys were homogenized by homogenizer on ice. Lipids were extracted by adjusting the solvent mixture to give a final ratio of chloroform/methanol/water equal to 4:8:3. The extract was incubated at room temperature with end-over-end agitation. The insoluble proteinaceous material was collected by centrifugation and re-extracted three times. The final pellet of insoluble protein was further washed with cold-acetone/water (4:1, v/v) four times and dried under a stream of nitrogen. O-linked glycan preparation O-linked carbohydrate fractions were cleaved from protein rich powder by ?-elimination procedures. Briefly, 500 uL ~ 1 mL of 1 M sodiumborohydride in 50 mM Sodiumhydroxide (NaOH) were added to the samples and incubated overnight at 45oC. The incubated samples were neutralized with 10%acetic acid and desalted by passing through a packed column of dowexTM resins (50 W x 8--100, Sigma Aldrich, St. Louis,MO) and lyophilized. The borate was removed with methanol/acetic acid (9:1) under a streamof nitrogen gas, and the samples were passed through a C18 reversed phase cartridge. The carbohydrate fractions (O-linked glycans) were eluted with 5% acetic acid. The carbohydrate fractions were 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 NSI-MSn analysis was performed following the method developed at the Complex Carbohydrates Research Center (Aoki et. al, 2007). Mass analysis was determined by using on a LTQ Orbitrap XL mass spectrometer (ThermoFisher) equipped with a nanospray ion source. Permethylated glycans were dissolved in 1mM NaOH in 50% methanol and infused directly into the instrument at a constant flow rate of 0.5 ?L/ min. A full FTMS spectrum was collected at 30 000 resolution. The capillary temperature was set at 210oC and MS analysis was performed in the positive ion mode. For total ion mapping (automated MS/MS analysis), m/z range, 800 to 2000 was scanned with ITMS mode in successive 2.8 mass unit windows that overlapped the preceding window by 2 mass units.
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