OLIGOSACCHARIDE PROFILING OF HUMAN MILK FRACTION 2

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. Preparation of milk samples and HPLC fractionation The 4 tubes of milk samples were spun in a centrifuge and 6 mL of the supernatant from each tube was filtered through a tightly packed glass wool into a 50-mL conical tube, lyophilized, and weighed. The dried sample was dissolved in nanopure H2O to obtain a concentration of 100 mg/mL. The sample was not pre-fractionated through a C18 sep pak cartridge to avoid exposure to room temperature for a certain period of time. About 100 [unreadable]L of the solution was injected into a HPLC machine 10 times and ten (10) fractions were collected across each run cycle. Similar fractions were composited from all 10 injection times. Fraction collection times and gradient program are presented on the graph of the first fraction collection. The milk solution was kept at refrigeration temperature (except at actual injection) and fractions (eluates) collected were kept frozen in dry ice at all injection times as composites were added because of concern of a change in composition profile. Gradient program, eluents, and column used were the same as in the previous progress report (Reference: Progress Report As of June 25, 2008;Analyst, Zhirui Wang). Milk oligosaccharides profiling by MALDI-TOF MS <Per-O-methylations of carbohydrates and purification by C18 sep-pak cartridge> About 50-uq aliguot of human milk fraction 2 (previously fractionated by HPLC: Reference Report EB091808Z dated October 29, 2008) was permethylated for oligosaccharide profiling (Ciucanu and Kerek, 1984). The aliquot was dissolved with dimethylsulfoxide and then methylated with NaOH and methyl iodide. The reaction was quenched by addition of water, and per-O-methylated carbohydrates were extracted with dichloromethane. Per-O-methylated glycans were cleaned of contaminants. Briefly, the glycans were dissolved in 1:1 methanol:water and loaded into a C18 sep pak cartridge and then washed with nanopure water. Per-O-methyl carbohydrated were eluted with 15% acetonitrile into a screw-cap tube, and with 85% acetonitrile into another screw-cap tube. The glycans eluted with 85% acetonitrile were dried under a stream of nitrogen gas and were dissolved with methanol for analysis by mass spectrometry. Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) MALDI-TOF-MS was performed in the reflector positive ion mode using a-dihyroxybenzoic acid (DHBA, 20 mg/mL in 50% methanol:water) as a matrix. Full mass spectrum of the sample was obtained by using a 4700 Proteomics analyzer (Applied Biosystems).