On-line LC-MS approach combining collision-induced dissociation (CID), electron-transfer dissociation (ETD), and CID of an isolated charge-reduced species for the trace-level characterization of proteins with post-translational modifications
On-line LC-MS approach combining collision-induced dissociation (CID), electron-transfer dissociation (ETD), and CID of an isolated charge-reduced species for the trace-level characterization of proteins with post-translational modifications
复制标题
DOI：
10.1021/pr070313u
复制
发表时间：
2007-11-01
影响因子：
4.4
通讯作者：
Karger, Barry L.
中科院分区：
文献类型：
--
作者：
Wu, Shiaw-Lin;Huehmer, Andreas F. R.;Karger, Barry L.
我们已经扩展了我们最近用于大肽分析的在线LC-MS平台，以组合联合收割机碰撞诱导解离（CID）、电子转移解离（ETD）和衍生自ETD的分离的电荷减少（CRCID）物质的CID，以确定磷酸化和糖基化修饰的位点，以及大肽片段的序列（即，2000-10 000 Da），从复杂的蛋白质，如P-酪蛋白，表皮生长因子受体（EGFR），和组织纤溶酶原激活剂（t-PA）在低飞摩尔水平。当自动选择具有高m/z（大约> 1000）的前体离子用于碎裂时，结合用于从ETD碎片离子分离的电荷减少的物质的额外CID活化步骤改善了ETD碎裂。具体而言，通过肽序列与近连续产物离子系列的广泛覆盖来加强精确磷酸化位点的鉴定。通过增强糖基化前体的肽骨架序列的鉴定，还改进了EGFR中Winked糖基化位点和t-PA中O-连接糖基化位点的鉴定。新的策略是一个很好的开始调查扫描，以表征酶肽混合物在广泛的质量范围内使用LC-MS与数据依赖性采集，因为三个激活步骤可以提供互补的信息给对方。一般而言，大肽可以通过ETD和CRCID步骤进行广泛表征，包括来自生成的近连续产物离子系列的修饰位点，并通过CID-MS 2步骤进行补充。同时，小肽（例如，
We have expanded our recent on-line LC-MS platform for large peptide analysis to combine collision-induced dissociation (CID), electron-transfer dissociation (ETD), and CID of an isolated charge-reduced (CRCID) species derived from ETD to determine sites of phosphorylation and glycosylation modifications, as well as the sequence of large peptide fragments (i.e., 2000-10 000 Da) from complex proteins, such as P-casein, epidermal growth factor receptor (EGFR), and tissue plasminogen activator (t-PA) at the low femtomol level. The incorporation of an additional CID activation step for a charge-reduced species, isolated from ETD fragment ions, improved ETD fragmentation when precursor ions with high m/z (approximately > 1000) were automatically selected for fragmentation. Specifically, the identification of the exact phosphorylation sites was strengthened by the extensive coverage of the peptide sequence with a near-continuous product ion series. The identification of Winked glycosylation sites in EGFR and an O-linked glycosylation site in t-PA were also improved through the enhanced identification of the peptide backbone sequence of the glycosylated precursors. The new strategy is a good starting survey scan to characterize enzymatic peptide mixtures over a broad range of masses using LC-MS with data-dependent acquisition, as the three activation steps can provide complementary information to each other. In general, large peptides can be extensively characterized by the ETD and CRCID steps, including sites of modification from the generated, near-continuous product ion series, supplemented by the CID-MS2 step. At the same time, small peptides (e.g.,