O-LINKED OLIGOSACCHARIDE PROFILING OF ONE SAMPLE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. First of all, the glycoprotein sample in IEF gel pieces was digested and extracted from gel pieces following the method of Giorgianni et al. 2003 (Electrophoresis 2003, 24, 253259) and then O-glycans were released from the tryptic digest by b-elimination procedure. The reaction mixture was further desalted, cleaned of borate and released O-glycans were separated from residual peptides by passing though C18 sep-pak cartridge. The O-glycans thus obtained were permethylated and profiled by mass spectrometry. The detailed procedures are shown below. In-gel digestion IEF gel slices were cut into smaller pieces (~3 mm3) and washed with 85% Acetonitrile three times. Dehydrated gels were reswelled with trypsin solution (trypsin in 40 mM Ammonium bicarbonate, AmBic) on ice for 45 min initially, and protein digestion was carried out at 37¿ C overnight. Subsequently, the supernatant was transferred into another tube. Peptides and the glycopeptides were extracted from the gel in series with 20% acetonitrile in 5% formic acid, 50% acetonitrile in 5% formic acid and then 80% acetonitrile in 5% formic acid. The peptides/glycopeptides extracts were dried and combined into one glass tube. O-linked glycan preparation O-linked carbohydrate fractions were cleaved from the tryptic digests by ¿-elimination procedures. Briefly, 500 ¿L of 50 mM Sodium hydroxide (NaOH) containing 19 mg of sodium borohydride were added to the samples and incubated overnight at 45o C. The incubated samples then were neutralized with 10% acetic acid and desalted by passing through a packed column of DOWEXTM resins (50W x 8  100, Sigma Aldrich) and then were lyophilized. Dried samples were cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas. Then the samples were further passed through a C18 reversed phase cartridge to purify the O-glycans. O-linked glycans were eluted with 5% acetic acid, lyophilized and permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) and profiled by mass spectrometry. Matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) 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. All spectra were obtained by using a 4700 Proteomics analyzer (Applied Biosystems). NanoSpray ionization-Linear Ion Trap Mass Spectrometry (LTQ) Mass spectrometric analysis was performed following the method developed at the Complex Carbohydrate Research Center (Aoki K, Perlman M, Lim JM, Cantu R, Wells L, Tiemeyer M. J Biol Chem. 2007 Mar 23;282(12):9127-42.). Mass analysis was determined by using NSI-LTQ/MSn. Briefly, permethylated glycans were dissolved in 1mM NaOH in 50% methanol and infused directly into the instrument (LTQ, Thermo Finnigan) at a constant flow rate of 0.4 ¿L/min. 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 (at 28 collision energy), m/z range, 500 to 2000 was scanned in successive 2.8 mass unit windows that overlapped the preceding window by 2 mass units.