School of Biotechnology and Biomolecular Sciences , University of New South Wales , Sydney , New South Wales 2052 , Australia.
School of Chemistry , University of New South Wales , Sydney , New South Wales 2052 , Australia.
Anal Chem. 2019 Sep 17;91(18):11827-11833. doi: 10.1021/acs.analchem.9b02504. Epub 2019 Aug 30.
Methylation of arginine residues in proteins, an enzyme-mediated post-translational modification (PTM), is important for mRNA processing and transport and for the regulation of many protein-protein interactions. However, proteolytic peptides resulting from alternative sites of post-translational methylation have identical masses and cannot be readily separated by standard liquid chromatography-mass spectrometry. Unlike acetylation or phosphorylation, methylation of arginine does not strongly affect the charge states of peptide ions, multiple instances of methylation can occur on a single amino acid residue, and the relative mass of the modification is <1% that of the typical proteolytic peptide. High field asymmetric waveform ion mobility spectrometry (FAIMS) is an orthogonal separation method to liquid chromatography that can rapidly separate gaseous ions prior to detection by mass spectrometry. Here, we report that FAIMS can be used to separate arginine-methylated peptides that differ by the position of a single methyl group for both mono- and dimethylated variants. Although the resolution of separation for these arginine-methylated peptides improved with increasing amounts of helium in the FAIMS carrier gas as expected, we found that the site of methylation can strongly affect the dependence of the electric field used for ion transmission on the extent of helium in the carrier gas. Thus, certain isobaric peptides can be cotransmitted at high helium concentrations whereas lower concentrations can be used for successful separations of such peptide mixtures. The capability to rapidly resolve isobaric arginine-methylated peptides should be useful in the future for the detailed analysis of protein arginine methylation in biological samples.
蛋白质精氨酸残基的甲基化是一种酶介导的翻译后修饰（PTM），对于 mRNA 加工和运输以及许多蛋白质-蛋白质相互作用的调节非常重要。然而，由于翻译后甲基化的替代位点而产生的蛋白水解肽具有相同的质量，并且不能通过标准的液相色谱-质谱法轻易分离。与乙酰化或磷酸化不同，精氨酸的甲基化不会强烈影响肽离子的电荷状态，可以在单个氨基酸残基上发生多个甲基化实例，并且修饰的相对质量<典型蛋白水解肽的 1%。高场非对称波形离子淌度质谱（FAIMS）是一种与液相色谱正交的分离方法，可以在质谱检测之前快速分离气态离子。在这里，我们报告说 FAIMS 可用于分离精氨酸甲基化肽，这些肽在单个甲基化位置上的差异对于单甲基化和二甲基化变体都适用。尽管如预期的那样，随着 FAIMS 载气中氦气含量的增加，这些精氨酸甲基化肽的分离分辨率有所提高，但我们发现甲基化的位置会强烈影响用于离子传输的电场对载气中氦气程度的依赖性。因此，某些等电肽可以在高氦浓度下共同传输，而较低的浓度可以成功分离此类肽混合物。快速分辨等电精氨酸甲基化肽的能力将来应该有助于对生物样品中蛋白质精氨酸甲基化的详细分析。
