Unblocking the sink: improved CID-based analysis of phosphorylated peptides by enzymatic removal of the basic C-terminal residue.
Unblocking the sink: improved CID-based analysis of phosphorylated peptides by enzymatic removal of the basic C-terminal residue.
复制标题
解除障碍物：通过酶促去除碱性C末端残基，改善了基于CID的磷酸化肽的分析。
DOI：
10.1007/s13361-013-0770-2
复制
发表时间：
2014-02
影响因子：
3.2
通讯作者：
Eyers, Claire E.
中科院分区：
文献类型：
--
作者：
Lanucara, Francesco;Lee, Dave Chi Hoo;Eyers, Claire E.
关键词：
提出了一种改进离子阱中基于碰撞诱导解离（CID）的磷酸化多肽串联质谱（MS/MS）分析的一步酶促反应。羧肽酶-B（CBP-B）用于选择性地去除磷酸化胰蛋白酶/Lys-C肽的C-末端精氨酸或赖氨酸残基，然后通过具有Paul型离子阱的CID进行MS/MS分析。这种基本的C-末端残基的去除起到限制磷酸（H3 PO 4）的气相中性损失的程度，有利于诊断B和y离子的形成，如通过增加序列特异性产物离子的数量和相对强度所确定的。当H3 PO 4消除如此占主导地位以致于阻碍了肽序列上磷酸化位点的定位时，这种差异片段化特别有价值。CBP-B处理后CID产生的串联质谱数据质量的改善导致磷酸肽一级序列分配和磷酸化位点定位的置信度更高。还生成了更高的Mascot离子评分，结合更低的期望值和更高的δ评分，以提高对部位分配的信心; Ascore值也得到了改善。这些结果是合理的，根据公认的机制消除H3 PO 4后，低能量CID和洞察的因素支配所观察到的解离途径。我们预计这种方法将是实用的磷酸化肽的MS分析，特别是当替代电子驱动的碎片技术不可用时。本文的在线版本（doi：10.1007/s13361-013-0770-2）包含补充材料，可供授权用户使用。
A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H3PO4), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H3PO4 elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H3PO4 upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available. ᅟ The online version of this article (doi:10.1007/s13361-013-0770-2) contains supplementary material, which is available to authorized users.
登录
查看更多内容
影响因子：
46.9
作者：
Knight, ZA;Schilling, B;Shokat, KM
通讯作者：
Shokat, KM
影响因子：
7
作者：
Marcantonio, Maria;Trost, Matthias;Thibault, Pierre
通讯作者：
Thibault, Pierre
DOI：
10.1016/s1044-0305(98)00088-9
发表时间：
1998-11-01
影响因子：
3.2
作者：
DeGnore, JP;Qin, J
通讯作者：
Qin, J
DOI：
10.1073/pnas.0611217104
发表时间：
2007-02-13
影响因子：
11.1
作者：
Molina, Henrik;Horn, David M.;Pandey, Akhilesh
通讯作者：
Pandey, Akhilesh
影响因子：
1.8
作者：
Chawner, Ross;Eyers, Claire E.;Gaskell, Simon J.
通讯作者：
Gaskell, Simon J.