IN-GEL DIGESTION AND O-LINKED OLIGOSACCHARIDE PROFILING OF TWO SAMPLES

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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. In-gel digestion, Peptide extraction from the gel The gel pieces first were cut into smaller pieces and then washed with 40mM AmBic. After 10 minutes, Ambic was removed and then the gel pieces were dehydrated with 100% acetonitrile for 10 minutes. The preceding washing steps were repeated until the gel turned white. After destaining, the gel pieces were reswelled in 10mM DTT in 40mM Ambic at 55¿ C for 1 hr. After cooling to room temperature, DTT solution was exchanged with 55mM IDA solution and then incubated in the dark for 45 minutes, and then gel pieces were washed with 40mM AmBic ( for 10 min) and exchanged with 100% acetonitrile (for 10 min). This second washing steps were repeated once. After drying with acetonitrile, the gel pieces were reswelled on ice with the trypsin solution (10ul of trypsin in 1ml of 40mM Ambic) for 45 minutes and then the proteins in the gel pieces were digested overnight at 37¿ C. The supernatant were transferred to a new tube and then the peptides and the glycopeptides were extracted from the gel pieces with 20% acetonitrile in 5% formic acid, 50% acetonitrile in 5% formic acid and then 80% acetonitrile in 5% formic acid. The sample solutions were dried and then reconstituted with nanopure water to composite them into one tube eventually. ¿- elimination, Desalting, Borate removal O-linked carbohydrate fractions were cleaved from the O-linked glycopeptides by ¿-elimination procedures. Briefly, 250 ¿L of 50 mM NaOH were added to each of the samples and then checked for pH. Upon determination that the pH was basic, another 250 ¿L of 50 mM NaOH containing 19 mg of sodium borohydride were added to the samples, vortexed, and incubated overnight at 450C. 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 before permethylation. Preparation of the per-O-methylated carbohydrates, cleaning up by C18 The lyophilized carbohydrate fraction was dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide (Ciucanu and Kerek, 1984). The reaction was quenched by addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. Per- O-methylated glycans were further cleaned of contaminants. Briefly, the glycans were loaded into a C18 sep pak cartridge and then washed with nanopure water and 15% acetonitrile. The glycans then were eluted with 85% acetonitrile. Purified glycans were dried under a stream of nitrogen gas and were dissolved with methanol prior to analysis by mass spectrometry. Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF) 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 Carbohydrates Research Center (Aoki K, Perlman M, Lim JM, Cantu R, Wells L, Tiemeyer M. J Biol Chem. 2007 Mar 23;282(12):9127-42. Epub 2007 Jan 29). 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. The collision energy was set at 28 for MS/MS fragmentation. For total ion mapping, automated MS/MS analysis (at 28 collision energy), m/z range from 500 to 2000 was scanned in successive 2.8 mass unit windows that overlapped the preceeding window by 2 mass units.