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. Summary of protocol The N-glycans were released and removed prior to b-elimination to avoid being mixed with the O-glycans in the latter's analysis. Briefly, glycoroteins were denatured and treated with trypsin and PNGase F. The released N-glycans were separated from the residual peptides and O-linked glycopeptides by passing the enzymatic digests through C18 sep pak cartridge. On the other hand, the O-glycans were released from the glycopeptides by ?- elimination. After ?- elimination, the samples were desalted and cleaned of borate, permethylated and analyzed by MALDI-TOF-TOF and ESI-LCQ mass spectrometry. Release of N-linked glycans The samples were dissolved with 50 mM ammonium bicarbonate and denatured by heating for 5 minutes at 100?C. After cooling to room temperature, the samples were digested with trypsin at 37oC, overnight. The samples then were heated at 100? C for 5 minutes to de-activate trypsin. A second enzyme, PNGase F (New England BioLabs) was added to the samples subsequently to release the N-glycans. After enzymatic digestions, the samples were passed through a C18 reversed phase cartridge. The carbohydrate fraction (N-linked glycans) was eluted first with 5% acetic acid, and the O-linked glycopeptides and peptides fraction was eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol into another screw-cap tube. The carbohydrate fraction was lyophilized immediately and stored, whereas the isopropanol fractions were evaporated initially under a stream of nitrogen and then lyophilized. Release of O-linked glycans by ?-elimination O-linked carbohydrates were cleaved from the glycopeptides by ?-elimination procedure. 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, desalted by passing through a packed column of Dowex resins, and lyophilized. Dried samples were cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas before permethylation. Preparation and purification of per-O-methylated carbohydrates The released O-glycans from each sample were dissolved in dimethyl sulfoxide 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. The glycans then were eluted with 85% acetonitrile. Purified glycans were dried under a stream of nitrogen gas and redissolved with methanol prior to analysis by MALDI and ESI mass spectrometry. Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) MALDI-MS was performed in the positive ion mode using ?-dihyroxybenzoic acid (DHBA, 20 mg/mL in 50% methanol:water) as a matrix. Full mass spectrum of each sample was obtained by using a 4700 Proteomics analyzer (Applied Biosystems). Profiling by ElectroSpray Ionization ?Linear Ion Trap mass Spectrometry (ESI-LCQ/MSn) The oligosaccharides detected by MALDI-TOF MS were confirmed by LCQ-ESI mass spectrometry. The remainder of the per-O-methylated O-linked glycans profiled through MALDI-TOF machine was diluted in 1 mM NaOH in 50% methanol (~5 pmol/?L) and infused directly into the LCQ-Advantage (Thermo Finnigan) instrument at a constant flow rate of 1.0 ?L/min via a syringe pump (Harvard Apparatus) and sprayed at 3.5 kV. A normalized collision energy of 35 and an isolation mass window of 2 Da was applied to obtain MSn.
Showing the most recent 10 out of 104 publications
