COLLISIONAL FRAGMENTATION OF GLYCOPEPTIDES BY ELECTROSPRAY IONIZATION LC MS AND LC MS MS - METHODS FOR SELECTIVE DETECTION OF GLYCOPEPTIDES IN PROTEIN DIGESTS
COLLISIONAL FRAGMENTATION OF GLYCOPEPTIDES BY ELECTROSPRAY IONIZATION LC MS AND LC MS MS - METHODS FOR SELECTIVE DETECTION OF GLYCOPEPTIDES IN PROTEIN DIGESTS
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DOI：
10.1021/ac00055a009
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发表时间：
1993-04-01
影响因子：
7.4
通讯作者：
CARR, SA
中科院分区：
文献类型：
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作者：
HUDDLESTON, MJ;BEAN, MF;CARR, SA
糖肽特异性检测的液相色谱/电喷雾质谱（LC/ESMS）的糖蛋白酶的质谱方法进行了探索，使用各种糖肽模型，然后应用于可溶性补体受体I型，一个240 kDa的糖蛋白含有25个潜在的N-糖基化位点。最具体的方法，需要一个三重四极杆，涉及监测糖oxonium片段离子在电子捕获器离子扫描ESMS/MS。来自非糖基化肽的信号几乎被消除，导致在总离子电流色谱痕量的糖肽存在于消化。相应的质谱产生分子量和糖肽微观异质性信息。我们称之为碰撞激发扫描的替代和补充方法也涉及将糖肽片段化为糖氧鎓离子片段，但不涉及任何质量选择过程，从而允许在单个四极杆仪器上进行实验。所得总离子色谱图与UV色谱图（215 nm）相似，但碳水化合物特异性离子（如N-乙酰己糖胺氧鎓离子（m/z 204））的选择离子色谱图产生糖肽特异性痕量。尽管在所示的含糖肽色谱峰的光谱中有时可能存在肽干扰，但后一种方法允许对同样指示糖肽在色谱图中位置的相同数据集进行肽图谱分析。 这两种方法都适用于检测所有常见类别的寡糖与肽的N-或O-连接的糖肽。
Mass spectrometric methods of glycopeptide-specific detection in liquid chromatography/electrospray mass spectrometry (LC/ESMS) of glycoprotein digests are explored using a variety of glycopeptide models and then applied to soluble complement receptor type I, a 240-kDa glycoprotein containing 25 potential sites of N-glycosylation. The most specific method, requiring a triple quadrupole, involves monitoring of sugar oxonium fragment ions during precursor-ion scan ESMS/MS. Signals derived from nonglycosylated peptides are virtually eliminated, resulting in a total-ion current chromatographic trace of only the glycopeptides present in the digest. The corresponding mass spectra yield molecular weight and glycopeptide microheterogeneity information. An alternative and complementary approach that we term collisional-excitation scanning also involves fragmentation of glycopeptides to sugar oxonium ion fragments but does not involve any mass-selection process, permitting the experiment to be performed on a single quadrupole Instrument. The resulting total ion chromatogram is similar to the UV chromatogram (215 nm), but a selected-ion chromatogram for carbohydrate-specific ions such as the N-acetylhexosamine oxonium ion (m/z 204) produces a glycopeptide-specific trace. Although there can sometimes be peptide interferences in the spectra of the indicated glycopeptide-containing chromatographic peaks, this latter approach permits peptide mapping to be performed on the same data set that also indicates the location of glycopeptides In the chromatogram. Both methods are suitable for detection of glycopeptides with all common classes of oligosaccharides in either N- or O-linkage to the peptide.