Metzger S, Hoffmann R
Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universität, Düsseldorf, Germany.
J Mass Spectrom. 2000 Oct;35(10):1165-77. doi: 10.1002/1096-9888(200010)35:10<1165::AID-JMS44>3.0.CO;2-R.
Phosphorylation of tyrosine residues in proteins is a common regulatory mechanism, although it accounts for less than 1% of the total O-phosphate content in proteins. Whereas aromatic phosphorylation sites can be identified by a number of different analytical techniques, sequence analysis of phosphotyrosine-containing proteins at the low picomole or even femtomole level is still a challenging task. This paper describes the post-source decay in matrix-assisted laser desorption/ionization mass spectrometry of phosphotyrosine-containing model peptides by comparing their fragmentation behavior with sequence-homologous unphosphorylated peptides. Whereas the parent ions showed significant losses of HPO3, all phosphorylated fragment ions of the b- and y-series displayed only minor dephosphorylated signals, which often were not detectable. Surprisingly, one of the studied phosphotyrosine-containing sequences displayed, in addition to the [M + H - 80]+ ion, a more abundant [M + H - 98]+ ion, which could be explained by elimination of phosphoric acid. This dephosphorylation pattern was very similar to the patterns obtained for phosphoserine- and phosphothreonine-containing peptides. Because the dephosphorylation pattern of the parent ion is often used to identify modified amino acids in peptides, we investigated possible dephosphorylation mechanisms in detail. Therefore, we substituted single trifunctional amino acid residues and incorporated deuterated phosphotyrosine residues. After excluding direct elimination of phosphoric acid from tyrosine, we could show that the obtained loss of H3PO4 depends on aspartic acid and arginine residues. Most likely the HPO3 group is transferred to aspartic acid followed by cleavage of phosphoric acid forming a succinimide. On the other hand, arginine appears to induce the H3PO4 loss by protonation of phosphotyrosine leaving a phenyl cation.
蛋白质中酪氨酸残基的磷酸化是一种常见的调节机制，尽管它在蛋白质中总的O - 磷酸含量中占比不到1%。虽然可以通过多种不同的分析技术鉴定芳香族磷酸化位点，但在低皮摩尔甚至飞摩尔水平对含磷酸酪氨酸的蛋白质进行序列分析仍然是一项具有挑战性的任务。本文通过比较含磷酸酪氨酸的模型肽与序列同源的未磷酸化肽的碎片化行为，描述了基质辅助激光解吸/电离质谱中的源后衰变。虽然母离子显示出显著的HPO₃损失，但b系列和y系列的所有磷酸化碎片离子仅显示出少量的去磷酸化信号，这些信号通常无法检测到。令人惊讶的是，除了[M + H - 80]+离子外，其中一个研究的含磷酸酪氨酸序列还显示出更丰富的[M + H - 98]+离子，这可以通过磷酸的消除来解释。这种去磷酸化模式与含磷酸丝氨酸和磷酸苏氨酸的肽所获得的模式非常相似。由于母离子的去磷酸化模式通常用于鉴定肽中的修饰氨基酸，我们详细研究了可能的去磷酸化机制。因此，我们替换了单个三官能氨基酸残基并掺入了氘代磷酸酪氨酸残基。在排除酪氨酸直接消除磷酸后，我们可以表明所获得的H₃PO₄损失取决于天冬氨酸和精氨酸残基。最有可能的是，HPO₃基团转移到天冬氨酸上，随后磷酸裂解形成琥珀酰亚胺。另一方面，精氨酸似乎通过磷酸酪氨酸的质子化诱导H₃PO₄损失，留下苯基阳离子。
