Gooley A A, Pisano A, Packer N H, Ball M, Jones A, Alewood P F, Redmond J W, Williams K L
Macquarie University Centre for Analytical Biotechnology (MUCAB), Sydney, NSW, Australia.
Glycoconj J. 1994 Jun;11(3):180-6. doi: 10.1007/BF00731216.
The characterization of site-specific glycosylation is traditionally dependent on the availability of suitable proteolytic cleavage sites between each glycosylated residue, so that peptides containing individual glycosylation sites are recovered. In the case of heavily glycosylated domains such as the O-glycosylated mucins, which have no available protease sites, this approach is not possible. Here we introduce a new method to gain site-specific compositional data on the oligosaccharides attached to a single amino acid. Using a model glycopeptide from a mutant human albumin Casebrook, glycosylated PTH-Asn was recovered after sequential solid-phase Edman degradation, subjected to acid hydrolysis and the sugars were identified by high performance anion exchange chromatography with pulsed amperometric detection. The PTH-Asn(Sac) derivative was further characterized by ionspray mass spectrometry. Comparison between an endoproteinase Glu-C glycopeptide and a tryptic glycopeptide showed that the oligosaccharide attached to Asn494 was stable after at least 10 cycles of Edman degradation.
位点特异性糖基化的表征传统上依赖于每个糖基化残基之间是否存在合适的蛋白水解切割位点，以便回收包含单个糖基化位点的肽段。对于像O-糖基化粘蛋白这样糖基化程度很高且没有可用蛋白酶切割位点的结构域，这种方法是不可行的。在此，我们引入了一种新方法，以获取连接到单个氨基酸上的寡糖的位点特异性组成数据。使用来自突变型人白蛋白Casebrook的模型糖肽，在连续固相埃德曼降解后回收糖基化的PTH-Asn，进行酸水解，并通过高效阴离子交换色谱-脉冲安培检测法鉴定糖类。通过离子喷雾质谱对PTH-Asn(Sac)衍生物进行了进一步表征。内肽酶Glu-C糖肽和胰蛋白酶糖肽之间的比较表明，连接到Asn494的寡糖在至少10个循环的埃德曼降解后是稳定的。
