Gropengiesser Jan, Varadarajan Balamurugan T, Stephanowitz Heike, Krause Eberhard
Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125 Berlin, Germany.
J Mass Spectrom. 2009 May;44(5):821-31. doi: 10.1002/jms.1581.
Qualitative and quantitative analysis of post-translational protein modifications by mass spectrometry is often hampered by changes in the ionization/detection efficiencies caused by amino acid modifications. This paper reports a comprehensive study of the influence of phosphorylation and methylation on the responsiveness of peptides to matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. Using well-characterized synthetic peptide mixtures consisting of modified peptides and their unmodified analogs, relative ionization/detection efficiencies of phosphorylated, monomethylated, and dimethylated peptides were determined. Our results clearly confirm that the ion yields are generally lower and the signal intensities are reduced with phosphopeptides than with their nonphosphorylated analogs and that this has to be taken into account in MALDI and ESI mass spectrometry. However, the average reduction of ion yield caused by phosphorylation is more pronounced with MALDI than with ESI. The unpredictable impact of phosphorylation does not depend on the hydrophobicity and net charge of the peptide, indicating that reliable quantification of phosphorylation by mass spectrometry requires the use of internal standards. In contrast to phosphorylation, mono- and dimethylated peptides frequently exhibit increased signal intensities in MALDI mass spectrometry (MALDI-MS). Despite minor matrix-dependent variability, MALDI methods are well suited for the sensitive detection of dimethylated arginine and lysine peptides. Mono- and dimethylation of the arginine guanidino group did not significantly influence the ionization efficiency of peptides in ESI-MS.
通过质谱对翻译后蛋白质修饰进行定性和定量分析，常常因氨基酸修饰导致的电离/检测效率变化而受到阻碍。本文报告了一项关于磷酸化和甲基化对肽段对基质辅助激光解吸/电离（MALDI）和电喷雾电离（ESI）质谱响应影响的综合研究。使用由修饰肽段及其未修饰类似物组成的特征明确的合成肽混合物，测定了磷酸化、单甲基化和二甲基化肽段的相对电离/检测效率。我们的结果清楚地证实，与非磷酸化类似物相比，磷酸化肽段的离子产率通常较低，信号强度降低，这在MALDI和ESI质谱分析中必须予以考虑。然而，磷酸化导致的离子产率平均降低在MALDI中比在ESI中更明显。磷酸化的不可预测影响并不取决于肽段的疏水性和净电荷，这表明通过质谱可靠地定量磷酸化需要使用内标。与磷酸化不同，单甲基化和二甲基化肽段在MALDI质谱（MALDI-MS）中经常表现出信号强度增加。尽管存在轻微的基质依赖性变化，但MALDI方法非常适合于灵敏检测二甲基化的精氨酸和赖氨酸肽段。精氨酸胍基的单甲基化和二甲基化在ESI-MS中对肽段的电离效率没有显著影响。
