Gehrig Peter M, Hunziker Peter E, Zahariev Sotir, Pongor Sándor
Institute of Biochemistry, University of Zurich, Zurich, Switzerland.
J Am Soc Mass Spectrom. 2004 Feb;15(2):142-9. doi: 10.1016/j.jasms.2003.10.002.
Protein methylation at arginine residues is a prevalent posttranslational modification in eukaryotic cells that has been implicated in processes from RNA-binding and transporting to protein sorting and transcription activation. Three main forms of methylarginine have been identified: N(G)-monomethylarginine (MMA), asymmetric N(G),N(G)-dimethylarginine (aDMA), and symmetric N(G),N'(G)-dimethylarginine (sDMA). To investigate gas-phase fragmentations and characteristic ions arising from methylated and unmodified arginine residues in detail, we subjected peptides containing these residues to electrospray triple-quadrupole tandem mass spectrometry. A variety of low mass ions including (methylated) ammonium, carbodiimidium, and guanidinium ions were observed. Fragment ions resulting from the loss of the corresponding neutral fragments (amines, carbodiimide, and guanidine) from intact molecular ions as well as from N- and C-terminal fragment ions were also identified. Furthermore, the peptides containing either methylated or unmodified arginines gave rise to abundant fragment ions at m/z 70, 112, and 115, for which cyclic ion structures are proposed. Electrospray ionization tandem mass spectra revealed that dimethylammonium (m/z 46) is a specific marker ion for aDMA. A precursor ion scanning method utilizing this fragment ion was developed, which allowed sensitive and specific detection of aDMA-containing peptides even in the presence of a five-fold excess of phosphorylase B digest. Interestingly, regular matrix-assisted laser desorption/ionization mass spectra recorded from aDMA- or sDMA-containing peptides showed metastable fragment ions resulting from cleavages of the arginine side chains. The neutral losses of mono- and dimethylamines permit the differentiation between aDMA and sDMA.
精氨酸残基的蛋白质甲基化是真核细胞中一种普遍的翻译后修饰，它参与了从RNA结合与转运到蛋白质分选和转录激活等过程。已鉴定出三种主要形式的甲基精氨酸：N(G)-单甲基精氨酸（MMA）、不对称N(G),N(G)-二甲基精氨酸（aDMA）和对称N(G),N'(G)-二甲基精氨酸（sDMA）。为了详细研究甲基化和未甲基化精氨酸残基产生的气相碎裂和特征离子，我们对含有这些残基的肽进行了电喷雾三重四极杆串联质谱分析。观察到了多种低质量离子，包括（甲基化的）铵离子、碳二亚胺离子和胍离子。还鉴定了完整分子离子以及N端和C端碎片离子失去相应中性碎片（胺、碳二亚胺和胍）后产生的碎片离子。此外，含有甲基化或未甲基化精氨酸的肽在m/z 70、112和115处产生了丰富的碎片离子，为此提出了环状离子结构。电喷雾电离串联质谱显示，二甲基铵（m/z 46）是aDMA的特异性标记离子。利用该碎片离子开发了一种前体离子扫描方法，即使在存在五倍过量的磷酸化酶B消化物的情况下，也能灵敏且特异性地检测含aDMA的肽。有趣的是，从含aDMA或sDMA的肽记录的常规基质辅助激光解吸/电离质谱显示了精氨酸侧链裂解产生的亚稳碎片离子。单甲胺和二甲胺的中性丢失允许区分aDMA和sDMA。
