| Glycobiology is an important part of life science research in 21st century. To have in-depth study of the functions and regulatory mechanism of glycans in life activities, the primary task is to obtain and analyze glycome. Electrospray Ionization Mass Spectrometry (ESI-MS) is a powerful tool to analyze micro-scale glycomes, but the pretreatment of sample is necessary. In this paper, we established a pretreatment procedure for N-linked glycans released from glycoproteins in micro-scale bio-samples before ESI-MS analysis in view of the exsting problems.This study firstly used two common glycoproteins, bovine ribonuclease B and chicken albumin, as research materials, and had an initial investigation on several releasing, purifying and enriching N-linked glycans methods and obtained the following conclusions:1. N-linked glycans releasing:we chose sodium dodecyl sulfate (SDS) and iodine acetamide (DTT) as protein denaturing agents. Glycoproteins were dissolved in the buffer and denatured at 100℃for 10 min. Then added sodium pyrophosphate buffer,10% ethyl-phenyl-polyethyleneglycol (NP40), PNGase F,37℃24 h. This approach is effective, efficient and easy to operate.2. N-linked glycans purification and enrichment:the enzymatic samples were pretreated separately by five enrichment and purification methods used in conjunction before mass spectrometry analysis. The results showed, in addition to the method of using microcrystalline cellulose and cation exchange resin in tandem, the other four kinds (combination of microcrystalline cellulose and cation exchange resin column, combination of C18 column and graphite carbon column, combination of C18 column and cation exchange resin and combination of graphite carbon column and microcrystalline cellulose column) were able to achieve a satisfactory effect of purification and enrichment.Then, we used five bio-samples, fetus bovine serum, control mouse serum, control human serum, mouse HAbI8 IgG and HAbI8 IgG (ascites method) for further verification. Moreover, we improved this method focusing on micro-scale samples:3. PVDF membrane assisted N-linked glycans releasing:the PVDF membrane has the characteristic of absorbing proteins. We used it to roughly purify the complex bio-samples prior to PNGase F digestion, and then in accordance with the above steps completed glycoprotein denaturation and enzymatic digestion directly on the membrane. The results showed that this method can also improve the purification effect of glycans from micro-scale samples.4. Improved procedure of purifying and enriching.N-linked glycans:In the above mentioned four kinds of enrichment and purification methods, only the combination of graphite carbon column and microcrystalline cellulose column had the best purifying and enriching effect. We used 0.1% trifluoroacetic acid containing 50% acetonitrile solution as eluting agent of graphite carbon column to avoid eluting free glycans incompelely; the pretreatment time of microcrystalline cellulose powder was related with quantities, and the effect of pretreatment would have a direct impact on glycans analysis. The micro-scale samples only required a small amount of powder, so we made use of the 1000μL standard pipettes as the disposable chromatography columns and greatly reduced impurities, improved the efficiency of purification.This study, based on ESI-MS technology, established a new procedure which was more suitable for purifying and enriching N-linked glycans from micro-scale bio-samples glycoproteins--PVDF membrane assisted N-linked glycans releasing, purifying and enriching free glycans by graphite carbon column and microcrystalline cellulose column. This method was rapid, stable, versatile and appliable to high-throughput N-linked glycans production before ESI-MS analysis fromμg grade bio-samples glycoproteins and propitious to subsequent glycomic research.
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