MALDI/TOF-MS WITH RELEASE OF N-GLYCANS

University Of Georgia, Athens GA

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Abstract

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. Each sample was dissolved in 50mM ammonium bicarbonate, and then 1 ul of the solution was analyzed by MALDI. The left-over samples were treated with PNGase F and the released glycans were analyzed by MALDI. Furthermore, the released glycans from GC4-1 and GC4-2 were permethylated and analyzed by NSI-LTQ mass spectrometry to confirm the biantennary bisecting structure. The detailed procedures are shown below. Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF) MALDI/TOF-MS was performed in the reflector positive ion mode using [unreadable]-dihyroxybenzoic acid as a matrix (5mg DHBA/1ml of 30% Acetonitrile in 0.1% TFA for intact or digested N-glycosyl asparagine derivatives, 20mg DHBA/1ml of 50% Methanol for permethylated glycans). The spectrum was obtained by using a 4700 Proteomics analyzer (Applied Biosystems). Release of N-glycans from N-glycosyl asparagine derivatives Each sample was dried and reconstituted with 50 mM sodium phosphate buffer (pH 7.5) and incubated with PNGase F at 37[unreadable] C overnight to release N-glycans. An aliquot of the digest was examined by MALDI. Glycan preparation for oligosaccharide analysis The tryptic digest 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 MALDI/TOF and NSI-MSn mass spectrometry to confirm biantennary bisecting structure. NanoSpray ionization-Linear Ion Trap Mass Spectrometry (NSI-LTQ/MSn) Mass spectrometric analysis was performed based on 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 [unreadable]L/min. The capillary temperature was set at 210oC and MS analysis was performed in the positive ion mode. The exact conformation of the structure was elucidated by performing stepwise MSMS analysis (at 35 collision energy) through capturing the residual ion after fragmentation