Liu J, Volk K J, Kerns E H, Klohr S E, Lee M S, Rosenberg I E
Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT 06492.
J Chromatogr. 1993 Feb 19;632(1-2):45-56. doi: 10.1016/0021-9673(93)80024-3.
An in-house modified microcolumn liquid chromatography (LC) system has been coupled to a PE-SCIEX API III triple-quadrupole mass spectrometer through an ionspray interface for the structural characterization of model glycoproteins, bovine ribonuclease B and human alpha 1-acid glycoprotein. In conjunction with enzymatic digestion approaches using trypsin and peptide-N-glycosidase F, the feasibility of packed-capillary (250 microns I.D.) LC columns, coupled with ionspray mass spectrometry (MS) in a tandem format, have been assessed for glycopeptide mapping and structural determination. This configuration demonstrates a highly promising approach for the determination of glycosylation sites and the corresponding sequence structures of related tryptic fragments. A glycosylated tetrapeptide, Asn-Leu-Thr-Lys with carbohydrate moieties on Asn-34, was readily located for bovine ribonuclease B. Preliminary results using micro-LC-MS also show the identification of a class A carbohydrate attachment on a tryptic fragment of human alpha 1-acid glycoprotein. The microheterogeneity of carbohydrate moieties can be quickly screened using this approach for either tryptic digests or the intact glycoprotein. These methods demonstrate potential applications for structural characterization of recombinant glycoproteins of pharmaceutical interest.
一种内部改良的微柱液相色谱（LC）系统已通过离子喷雾接口与PE-SCIEX API III三重四极杆质谱仪联用，用于对模型糖蛋白、牛核糖核酸酶B和人α1-酸性糖蛋白进行结构表征。结合使用胰蛋白酶和肽-N-糖苷酶F的酶解方法，已评估了内径为250微米的填充毛细管LC柱与串联形式的离子喷雾质谱（MS）联用进行糖肽图谱分析和结构测定的可行性。这种配置展示了一种极具前景的方法，可用于确定糖基化位点以及相关胰蛋白酶片段的相应序列结构。对于牛核糖核酸酶B，很容易定位到一个糖基化四肽，即Asn-34上带有碳水化合物部分的Asn-Leu-Thr-Lys。使用微LC-MS的初步结果还显示，在人α1-酸性糖蛋白的一个胰蛋白酶片段上鉴定出了A类碳水化合物连接。使用这种方法可以快速筛选胰蛋白酶消化产物或完整糖蛋白中碳水化合物部分的微异质性。这些方法展示了在对具有药学意义的重组糖蛋白进行结构表征方面的潜在应用。
