UTILITY OF NONCOVALENT COMPLEXES IN THE MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY OF HEPARIN-DERIVED OLIGOSACCHARIDES
UTILITY OF NONCOVALENT COMPLEXES IN THE MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY OF HEPARIN-DERIVED OLIGOSACCHARIDES
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DOI：
10.1016/0008-6215(94)00012-5
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发表时间：
1995-04-30
影响因子：
3.1
通讯作者：
BIEMANN, K
中科院分区：
文献类型：
--
作者：
JUHASZ, P;BIEMANN, K
肝素衍生的寡糖的分子量范围从二糖到十六糖已确定由基质辅助激光解吸电离飞行时间质谱。虽然这些化合物在原样使用时吸收差或根本不吸收，但可以获得它们与碱性肽或蛋白质形成的离子复合物的强信号。硫酸化低聚糖的分子量通过从复合物的质量中减去碱性组分的质量来确定。实验条件的优化导致亚皮摩尔的灵敏度，在消除硫酸盐损失和干扰的无机阳离子的附件。合成肽（Arg-Gly）（10）和（Arg-Gly）（10）被专门设计为合成和天然肝素片段直至十糖的络合剂。化学均质寡糖（+/-0.05%）的精确分子量测定明确鉴定了糖单元的数量以及O、N-硫酸酯和N-乙酰基的数量。对于大于十糖的寡糖，使用小的碱性蛋白质血管生成素（M（r）= 14，120）来形成复合物（不均匀的十六糖部分是本研究可获得的最大部分）。对于大于十糖的不均匀样品，质量准确度较低（+/-0.2-0.3%），但仍足以测定存在的糖单元数并估计硫酸根基团数，只是不再可能区分一个硫酸根和两个N-乙酰基（Delta = 4 Da）。然而，考虑到已知的硫酸化和乙酰化，肝素裂解酶和化学降解步骤的特异性，该方法有望显着有助于确定肝素和其他硫酸化糖胺聚糖的一级结构。
Molecular weights of heparin-derived oligosaccharides ranging from disaccharides to hexadecasaccharides have been determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. While these compounds ionize poorly or not at all when used as such, a strong signal can be obtained of their ionic complexes formed with a basic peptide or protein. The molecular weight of the sulfated oligosaccharide is determined by subtracting the mass of the basic component from that of the complex. Optimization of the experimental conditions resulted in sub-picomole sensitivity, in the elimination of sulfate loss and of the interference from attachment of inorganic cations. Synthetic peptides (Arg-Gly)(10) and (Arg-Gly)(10) were specifically designed as complexing agents for synthetic and natural heparin fragments up to decasaccharides. Accurate molecular weight determination on chemically homogeneous oligosaccharides (+/-0.05%) unambiguously identified the number of saccharide units, and the number of O,N-sulfate and N-acetyl groups. For oligosaccharides larger than decasaccharides, a small basic protein, angiogenin (M(r) = 14,120), was used to form the complex (an inhomogeneous hexadecasaccharide fraction was the largest available for this study). For inhomogeneous samples larger than decasaccharides, the mass accuracy is lower (+/-0.2-0.3%) but still suffices to determine the number of saccharide units present and to estimate the number of sulfate groups, except it is no longer possible to differentiate one sulfate from two N-acetyl groups (Delta = 4 Da). However, taking into account known regularities of sulfation and acetylation, the specificity of heparin lyases and chemical degradation steps, the method promises to contribute significantly to the determination of the primary structure of heparin and other sulfated glycosaminoglycans.