Accurate localization and relative quantification of arginine methylation using nanoflow liquid chromatography coupled to electron transfer dissociation and orbitrap mass spectrometry.
Accurate localization and relative quantification of arginine methylation using nanoflow liquid chromatography coupled to electron transfer dissociation and orbitrap mass spectrometry.
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DOI：
10.1016/j.jasms.2008.11.008
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发表时间：
2009-03
影响因子：
3.2
通讯作者：
Qu J
中科院分区：
文献类型：
--
作者：
Wang H;Straubinger RM;Aletta JM;Cao J;Duan X;Yu H;Qu J
蛋白质精氨酸（Arg）甲基化在真核细胞中起着重要的功能作用，通常发生在由多个Arg组成的区域。甲基精氨酸（MA）在富精氨酸结构域的定位对基于质谱（MS）的方法提出了挑战；肽在电喷雾电离（ESI）作用下是高电荷的，这限制了碰撞诱导解离（CID）产生的序列信息产物的数量，并且在CID过程中不稳定甲基化部分的损失阻碍了肽主链的有效断裂。本文利用电子转移解离（ETD）和CID对甲基化和未修饰的甘氨酸/富含精氨酸肽（GAR）进行了全面的断裂行为研究，这些肽来源于人核蛋白残基679-695，其中包含在生物系统中广泛存在的甲基化基元。ETD为测序和MA定位提供了丰富的信息，而CID未能为任何可用的电荷状态提供可靠的识别（z=2-4）。然而，CID产生的特征中性损失可以用来区分不同类型的MA，正如之前的工作所建议的那样，并且在这里通过LTQ/Orbitrap的高精度/分辨率的产品离子扫描得到证实。为了分析相对复杂混合物中的MA肽，开发了一种方法，采用纳米lc耦合交替CID/ETD进行肽测序和MA定位/表征，并使用Orbitrap进行准确的前体测量和MA肽化学计量的相对定量。为了证明这一概念，我们分析了蛋白精氨酸n -甲基转移酶PRMT1和PRMT7在体外甲基化gar肽的情况。结果表明，PRMT1产生大量单甲基化（MMA）和不对称二甲基化肽，而PRMT7主要产生MMA肽和一些对称二甲基化肽。这种方法和结果可能会促进对PRMTs的作用和Arg甲基化模式的功能意义的理解。
Protein arginine (Arg) methylation serves an important functional role in eukaryotic cells, and typically occurs in domains consisting of multiple Arg in close proximity. Localization of methylarginine (MA) within Arg-rich domains poses a challenge for mass spectrometry (MS)-based methods; the peptides are highly-charged under electrospray ionization (ESI), which limits the number of sequence-informative products produced by collision induced dissociation (CID), and loss of the labile methylation moieties during CID precludes effective fragmentation of the peptide backbone. Here the fragmentation behavior of Arg-rich peptides was investigated comprehensively using electron transfer dissociation (ETD) and CID for both methylated and unmodified glycine-/Arg-rich peptides (GAR), derived from residues 679-695 of human nucleolin, which contains methylation motifs that are widely-represented in biological systems. ETD produced abundant information for sequencing and MA localization, whereas CID failed to provide credible identification for any available charge state (z=2-4). Nevertheless, CID produced characteristic neutral losses that can be employed to distinguish among different types of MA, as suggested by previous works and confirmed here with product ion scans of high accuracy/resolution by an LTQ/Orbitrap. To analyze MA-peptides in relatively complex mixtures, a method was developed that employs nano-LC coupled to alternating CID/ETD for peptide sequencing and MA localization/characterization, and an Orbitrap for accurate precursor measurement and relative quantification of MA-peptide stoichiometries. As proof of concept, GAR-peptides methylated in vitro by protein arginine N-methyltransferases PRMT1 and PRMT7 were analyzed. It was observed that PRMT1 generated a number of monomethylated (MMA) and asymmetric-dimethylated peptides, while PRMT7 produced predominantly MMA peptides and some symmetric-dimethylated peptides. This approach and the results may advance understanding of the actions of PRMTs and the functional significance of Arg methylation patterns.
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影响因子：
7.4
作者：
Diehnelt, CW;Dugan, NR;Budde, WL
通讯作者：
Budde, WL
影响因子：
7.4
作者：
Jung, Sung Yun;Li, Yehua;Qin, Jun
通讯作者：
Qin, Jun
影响因子：
4.8
作者：
Klein, S;Carroll, JA;Rifkin, DB
通讯作者：
Rifkin, DB
DOI：
10.1016/s1387-2656(08)00008-2
发表时间：
2008-01-01
期刊：
BIOTECHNOLOGY ANNUAL REVIEW, VOL 14
影响因子：
--
作者：
Aletta, John M.;Hu, John C.
通讯作者：
Hu, John C.
影响因子：
5.4
作者：
Lacovides, Demetris C.;O'Shea, Clodagh C.;McCormick, Frank
通讯作者：
McCormick, Frank