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, twelve coomassie blue stained gel slices (same samples are in the gel) were cut into smaller pieces and then combined in one tube. Then in-gel digestion was performed followed by extraction of glycopeptides, cleaned up by C18, PNGase F digestion, PNGase A digestion. The released N-glycans were separated from the residual peptides and O-linked glycopeptides by C18. The N-glycans were permethylated and analyzed by MALDI/TOF- MS and NSI-MSn. The procedures are shown in detail below. Coomassie blue-stained gel slices were cut into smaller pieces (~1 mm3) and destained alternately with 40mM Ammonium bicarbonate (AmBic) and 100% acetonitrile until the color turned clear. Destained gel was reswelled in 10 mM DTT in 40mM Ambic at 55? C for 1 hr. The DTT solution was exchanged with 55mM Iodoacetamide (IAM) and incubated in the dark for 45 min. Incubation was followed by washing alternately with 40mM AmBic and 100% acetonitrile twice. Dehydrated gel was reswelled with trypsin solution (trypsin in 140 mM Ambic) on ice for 45 min initially, and protein digestion was carried out at 37? C overnight. 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 sample solutions were dried and combined into one tube. Extracted tryptic digest was passed through a C18 sep-pak cartridge and washed with 5% acetic acid to remove contaminants (salts, SDS, etc.). Peptides and glycopeptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol and dried in a speed vacuum concentrator. The dried samples were combined and then reconstituted with 50 mM sodium phosphate buffer (pH 7.5) and heated at 100? C for 5 min to inactivate trypsin. The tryptic digest was incubated with PNGase F at 37? C overnight followed by PNGase A digestion with 200mM citrate buffer (pH 5.0) at 37oC overnight to release N-glycans. After digestion, the sample was passed through a C18 sep-pak cartridge and the carbohydrate fraction was eluted with 5% acetic acid and dried by lyophilization. Released N-linked oligosaccharides were permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) and profiled by 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. All spectra were obtained by using a 4700 Proteomics analyzer (Applied Biosystems). 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.). 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 30 collision energy), m/z range from 400 to 2000 was scanned in successive 2.8 mass unit windows that overlapped the preceeding window by 2 mass units.
