Although most peptide bonds in proteins exist in the trans configuration, when cis peptide bonds do occur, they can have major impact on protein structure and function. The rapid identification of cis peptide bonds is therefore an important task. Peptide bonds containing proline are more likely to adopt the cis configuration because the ring connecting the side chain and backbone in proline flattens the energetic landscape relative to amino acids with free side chains. Examples of cis proline isomers have been identified in both solution and in the gas phase by a variety of structure-probing methods. Mass spectrometry is an attractive potential method for identifying cis proline due to its speed and sensitivity; however, the question remains of whether cis/trans proline isomers originating in solution are preserved during ionization and manipulation within a mass spectrometer. Herein, we investigate the gas-phase stability of isolated solution-phase cis and trans proline isomers using a synthetic peptide sequence with a Tyr-Pro-Pro motif. A variety of dissociation methods were explored to evaluate their potential to distinguish cis/trans configuration, including collision-induced dissociation, radical-directed dissociation, and photodissociation. Only photodissociation employed in conjunction with extremely gentle electrospray and charge solvation by 18-crown-6 ether was able to distinguish cis/trans isomers for our model peptide, suggesting that any thermal activation during transfer or while in the gas phase leads to isomer scrambling. Furthermore, the necessity for 18-crown-6 suggests that intramolecular charge solvation taking place during electrospray ionization can override cis/trans isomer homogeneity. Overall, the results suggest that solution-phase cis/trans proline isomers are fragile and easily lost during electrospray, requiring careful selection of instrument parameters and consideration of charge solvation to prevent cis/trans scrambling.

中文翻译：

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用紫外光解离研究气相中脯氨酸的顺/反异构体的稳定性。

尽管蛋白质中的大多数肽键都以反式构型存在，但当确实出现顺式肽键时，它们可能对蛋白质的结构和功能产生重大影响。因此，快速鉴定顺式肽键是一项重要的任务。包含脯氨酸的肽键更可能采用顺式构型，因为连接脯氨酸侧链和主链的环相对于具有游离侧链的氨基酸使能量构象变平。顺式脯氨酸异构体的实例已通过多种结构探测方法在溶液和气相中得到鉴定。质谱法由于其速度和灵敏度而成为一种鉴定顺式脯氨酸的有潜力的方法。然而，仍然存在以下问题：在质谱仪中进行电离和操作期间，是否保留了源自溶液的顺式/反式脯氨酸异构体。本文中，我们使用具有Tyr-Pro-Pro基序的合成肽序列研究了分离的溶液相顺式和反式脯氨酸异构体的气相稳定性。探索了多种解离方法以评估其区分顺式/反式构型的潜力，包括碰撞诱导的解离，自由基定向的解离和光解离。只有光解离结合极温和的电喷雾和18冠冕6醚的电荷溶剂化才能区分模型肽的顺式/反式异构体，这表明在转移过程中或在气相中的任何热活化都会导致异构体加扰。此外，18-crown-6的必要性表明，在电喷雾电离过程中发生的分子内电荷溶剂化可以超越顺式/反式异构体的均质性。总体而言，结果表明溶液相顺/反脯氨酸异构体易碎，在电喷雾过程中容易丢失，需要仔细选择仪器参数并考虑电荷溶剂化以防止顺/反加扰。