Structural analysis of the CHO‐derived interleukin‐4 by liquid‐chromatography/electrospray ionization mass spectrometry
Structural analysis of the CHO‐derived interleukin‐4 by liquid‐chromatography/electrospray ionization mass spectrometry
复制标题
通过液相色谱/电喷雾电离质谱法对 CHO 衍生的白介素-4 进行结构分析
DOI：
--
复制
发表时间：
1995
期刊：
影响因子：
--
通讯作者：
T. Covey
中科院分区：
文献类型：
--
作者：
A. Tsarbopoulos;B. Pramanik;T. L. Nagabhushan;T. Covey
电喷雾（ES）电离质谱分析CHO衍生的重组白细胞介素-4（IL-4）之前和之后的肽：N-糖苷酶F去糖基化。这证明不仅在推导所连接的碳水化合物组分的大小方面有用，而且在鉴定作为单唾液酸化和二唾液酸化复合型N-连接寡糖的主要糖型方面也有用。还检测到由含有具有更多延伸或更高分支的碳水化合物组分的糖型产生的额外信号。通过将所得肽混合物的蛋白水解消化和色谱分离与在线ES质谱检测相结合，进行CHO IL-4的进一步作图。比较分析CHO IL-4及其去糖基化产物的V8蛋白酶活性，发现Asn 38 N-糖基化位点。通过搜索所得的原始ES数据以寻找信号对来鉴定含糖肽的级分，所述信号对的m/z值因针对信号电荷状态调整的各种碳水化合物单元的质量而不同（例如，含NeuAc单元的糖肽的三重电荷ES信号的差异为97 u）。此外，ES质谱分析在提高孔口电位允许的糖肽的总离子电流色谱图中的快速定位，通过监测几个碳水化合物的特定片段离子。这种高纯度诱导的片段化是一种高灵敏度的方法，用于在色谱分离的组分中产生糖诊断离子，即使当糖肽和肽片段共洗脱时也是如此。
Electrospray (ES) ionization mass spectrometric analysis of CHO-derived recombinant interleukin-4 (IL-4) before and after deglycosylation with peptide: N-glycosidase F is described. That proved useful not only in deriving the size of the attached carbohydrate components but also in identifying the major glycoforms as the mono-and disialylated complex-type N-linked oligosaccharides. Additional signals arising from glycoforms containing carbohydrate components with more extended or higher branching were also detected. Further mapping of CHO IL-4 was carried out by combining proteolytic digestion and chromatographic separation of the resulting peptide mixture with on-line ES mass spectrometric detection. Comparative analysis of the V8 protease digests of CHO IL-4 and its deglycosylated product revealed the Asn38N-glycosylation site. Glycopeptide-containing fractions were identified by searching the resulting raw ES data for signal pairs whose m/z values differ by the mass of various carbohydrate units adjusted for the signal's charge state (e.g. 97 u difference for a triply charged ES signal of a glycopeptide containing NeuAc units). Furthermore, ES mass spectrometric analysis at elevated orifice potentials allowed the rapid location of the glycopeptides in the total ion current chromatogram by monitoring several carbohydrate-specific fragment ions. This high orifice-induced fragmentation is a highly sensitive method for generating sugar diagnostic ions in chromatographically separated components, even when glycopeptide and peptide fragments are co-eluting.
登录
查看更多内容
通过质谱法对重组可溶性 CD4 受体进行蛋白质和碳水化合物结构分析。
DOI：
--
发表时间：
1989
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Carr,SA;Hemling,ME;Folena-Wasserman,G;Sweet,RW;Anumula,K;Barr,JR;Huddleston,MJ;Taylor,P
通讯作者：
Taylor,P
影响因子：
7.4
作者：
HUDDLESTON, MJ;BEAN, MF;CARR, SA
通讯作者：
CARR, SA
影响因子：
2.9
作者：
Yazdanparast,R;Andrews,P;Smith,DL;Dixon,JE
通讯作者：
Dixon,JE
通过 252Cf 等离子体解吸质谱法进行肽和蛋白质图谱分析。
影响因子：
2.9
作者：
Tsarbopoulos,A;Becker,GW;Occolowitz,JL;Jardine,I
通讯作者：
Jardine,I
定位蛋白质中二硫键的策略。
影响因子：
--
作者：
Smith,DL;Zhou,ZR
通讯作者：
Zhou,ZR