Rappsilber Juri, Friesen Westley J, Paushkin Sergey, Dreyfuss Gideon, Mann Matthias
Center for Experimental Bioinformatics and Department of Biochemistry & Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark.
Anal Chem. 2003 Jul 1;75(13):3107-14. doi: 10.1021/ac026283q.
Dimethylation at arginine residues has been shown to be central in cellular processes such as signal transduction, transcription activation, and protein sorting. The two methyl groups are either placed symmetric or asymmetric on the zeta standing nitrogen atoms of the arginine side chain. Here, we introduce a novel method that enables the localization of dimethylarginine (DMA) residues in gel-separated proteins at a level of sensitivity of better than 1 pmol and that allows one to distinguish between the isomeric symmetric and asymmetric position of the methyl groups. The method utilizes two side-chain fragments of DMA, the dimethylammonium ion (m/z 46.06) and the dimethylcarbodiimidium ion (m/z 71.06), for positive ion mode precursor ion scanning. Dimethylcarbodiimidium ions (m/z 71.06) are produced by symmetric as well as asymmetric dimethylarginine but are observed more strongly for symmetric DMA. It is utilized here in the precursor of m/z 71 scan to indicate the presence of DMA in a peptide. The dimethylammonium ion (m/z 46.06) is specific for asymmetric DMA and is utilized here in the precursor of m/z 46 scan. The positive ion mode allows for the identification of the protein by peptide sequencing and simultaneous detection and localization of the modified residues. The analysis can be conducted on any mass spectrometer capable of precursor ion scanning. However, the high resolution of a quadrupole TOF instrument is beneficial to assign the accurate charge state of the often highly charged precursors. Using the precursor of m/z 71 scan, we found FUS/TLS and Sam68 to be DMA-containing proteins. We discovered at least 20 DMA sites in FUS/TLS. In MS/MS, we observed neutral loss of dimethylamine (m/z 45.05) from which it follows that the dimethylation in FUS/TLS is asymmetric. Monitoring in parallel the fragments m/z 46.06 and 71.06 in precursor ion scans and peptide sequencing, we identified at least nine asymmetric DMA modifications in Sam68. The parallel monitoring of fragments in precursor ion scans is a versatile tool to specify the nature of protein modifications in cases where a single fragment is not conclusive.
精氨酸残基的二甲基化已被证明在细胞过程如信号转导、转录激活和蛋白质分选等中起着核心作用。两个甲基要么对称地要么不对称地置于精氨酸侧链的ζ位氮原子上。在此，我们介绍一种新方法，该方法能够在凝胶分离的蛋白质中定位二甲基精氨酸（DMA）残基，灵敏度优于1皮摩尔，并且能够区分甲基的异构体对称和不对称位置。该方法利用DMA的两个侧链片段，即二甲基铵离子（m/z 46.06）和二甲基碳二亚胺离子（m/z 71.06），用于正离子模式前体离子扫描。二甲基碳二亚胺离子（m/z 71.06）由对称和不对称二甲基精氨酸产生，但对于对称DMA观察到的信号更强。在此，它用于m/z 71扫描的前体中以指示肽中DMA的存在。二甲基铵离子（m/z 46.06）对不对称DMA具有特异性，在此用于m/z 46扫描的前体中。正离子模式允许通过肽测序鉴定蛋白质，并同时检测和定位修饰的残基。该分析可以在任何能够进行前体离子扫描的质谱仪上进行。然而，四极杆飞行时间仪器的高分辨率有利于确定通常带高电荷的前体的准确电荷状态。使用m/z 71扫描的前体，我们发现FUS/TLS和Sam68是含DMA的蛋白质。我们在FUS/TLS中发现了至少20个DMA位点。在串联质谱中，我们观察到二甲胺（m/z 45.05）的中性丢失，由此可知FUS/TLS中的二甲基化是不对称的。在前体离子扫描和肽测序中并行监测片段m/z 46.06和71.06，我们在Sam68中鉴定出至少九个不对称DMA修饰。在前体离子扫描中并行监测片段是一种通用工具，可在单个片段不确定的情况下确定蛋白质修饰的性质。
