This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.Preparation of the per-O-methylated carbohydrates, cleaning up by C18The 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).
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