Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.
Chemistry. 2011 Jan 10;17(2):486-97. doi: 10.1002/chem.201002483. Epub 2010 Nov 30.
Tissue transglutaminase (tTGase) catalyzes both deamidation and transamidation of peptides and proteins by using a peptidyl glutamine as primary substrate. A precise consensus sequence for the enzyme is unknown and the ratio between deamidated and transamidated (or cross-linked) reaction products is highly substrate-dependent. Due to its overlapping body distribution with tTGase and ease of manipulation with tandem mass spectrometry, we used the neuropeptide substance P as a model to investigate the associated enzymatic kinetics and reaction products. Online liquid-chromatography Fourier-transform ion-cyclotron-resonance mass spectrometry (FT-ICR MS) combined with electron-capture dissociation (ECD) was employed to study the tTGase-induced modifications of substance P. A particular strength of ECD for peptide-enzyme reaction product monitoring is its ability to distinguish isomeric amino acids, for example, Glu and iso-Glu, by signature product ions. Our studies show that the primary reaction observed is deamidation, with the two consecutive glutamine residues converted sequentially into glutamate: first Gln(5) , and subsequently Gln(6) . We then applied ECD FT-ICR MS to identify the transamidation site on an enzymatically cross-linked peptide, which turned out to correspond to Gln(5) . Three populations of substance-P dimers were detected that differed by the number of deamidated Gln residues. The higher reactivity of Gln(5) over Gln(6) was further confirmed by cross-linking SP with monodansylcadaverine (MDC). Overall, our approach described herein is of a general importance for mapping both enzymatically induced post-translational protein modifications and cross-linking. Finally, in vitro Ca-signaling assays revealed that the main tTGase reaction product, the singly deamidated SP (RPKPEQFFGLM-NH(2) ), has increased agonist potency towards its natural receptor, thus confirming the biologically relevant role of deamidation.
组织转谷氨酰胺酶（tTGase）通过使用肽基谷氨酰胺作为主要底物来催化肽和蛋白质的脱酰胺和转酰胺（或交联）反应。酶的精确共识序列尚不清楚，并且脱酰胺和转酰胺（或交联）反应产物的比例高度依赖于底物。由于其与 tTGase 重叠的体分布以及与串联质谱联用的易于操作，我们使用神经肽物质 P 作为模型来研究相关的酶促动力学和反应产物。在线液-色谱傅里叶变换离子回旋共振质谱（FT-ICR MS）与电子俘获解离（ECD）相结合用于研究 tTGase 诱导的物质 P 修饰。ECD 用于监测肽-酶反应产物的一个特别优势是其能够通过特征产物离子区分异构氨基酸，例如 Glu 和 iso-Glu。我们的研究表明，观察到的主要反应是脱酰胺作用，两个连续的谷氨酰胺残基依次转化为谷氨酸：首先是 Gln(5)，然后是 Gln(6)。然后，我们将 ECD FT-ICR MS 应用于鉴定酶交联肽上的转酰胺化位点，结果发现该位点对应于 Gln(5)。检测到三种物质 P 二聚体，它们的区别在于脱酰胺 Gln 残基的数量。Gln(5)的反应性高于 Gln(6)，这一结果通过用单丹磺酰尸胺（MDC）交联 SP 进一步得到证实。总体而言，本文所述的方法对于映射酶诱导的翻译后蛋白质修饰和交联都具有重要意义。最后，体外 Ca 信号转导测定表明，主要的 tTGase 反应产物，即单脱酰胺物质 P（RPKPEQFFGLM-NH(2)），对其天然受体具有增加的激动剂效力，从而证实了脱酰胺作用的生物学相关性。
