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: First, lipids were extracted and proteins were precipitated from fermentation solution by adding chloroform and methanol to the solution. The precipitate was further washed with acetone/water solution four times. A small aliquot of protein rich powder (1%) was used for examine the protein powder by SDS-PAGE and the rest of the powder was subjected to b-elimination for O-glycan analysis. The solution after b-elimination was then desalted by passing though Dowex resin, followed by borate removal and C18 clean up. The released O-glycans thus obtained were permethylated and profiled by mass spectrometry. The detailed procedures used for your sample analysis are shown in detail below. Preparation of protein rich powder from fermentation solution Protein rich powder was prepared from fermentation solution according to the method of Aoki.et.al (2007). Briefly, lipids were removed and proteins were precipitated from the fermentation solution 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. SDS-PAGE An aliquot of protein rich powder (1%) were analyzed by SDS-PAGE to see protein content in protein rich powder. The samples were separated in a 4-15% SDS-PAGE gradient gel. After electrophoresis, the resolved proteins were stained with Coomassie blue. O-linked glycan preparation O-linked carbohydrate fractions were cleaved from protein rich powder by ?-elimination procedures. Briefly, 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 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 AB SCIEX TOF/TOF"""""""" 5800 System (Applied Biosystems). 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.
Showing the most recent 10 out of 104 publications
