Silzel Jacob W, Murphree Taylor A, Paranji Rajan K, Guttman Miklos M, Julian Ryan R
Department of Chemistry, University of California, 501 Big Springs Road, Riverside, California 92521, United States.
Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, United States.
J Am Soc Mass Spectrom. 2020 Sep 2;31(9):1974-1980. doi: 10.1021/jasms.0c00242. Epub 2020 Aug 18.
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.
尽管蛋白质中的大多数肽键以反式构型存在，但当顺式肽键确实出现时，它们会对蛋白质的结构和功能产生重大影响。因此，快速识别顺式肽键是一项重要任务。含有脯氨酸的肽键更有可能采取顺式构型，因为脯氨酸中连接侧链和主链的环相对于具有自由侧链的氨基酸而言，使能量格局变得平坦。通过多种结构探测方法，已在溶液和气相中鉴定出顺式脯氨酸异构体的实例。由于质谱分析速度快且灵敏度高，它是识别顺式脯氨酸的一种有吸引力的潜在方法；然而，源自溶液的顺式/反式脯氨酸异构体在质谱仪内的电离和操作过程中是否得以保留仍是个问题。在此，我们使用具有Tyr-Pro-Pro基序的合成肽序列，研究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它们区分顺式/反式构型的潜力，包括碰撞诱导解离、自由基导向解离和光解离。只有将光解离与极其温和的电喷雾以及18-冠-6醚的电荷溶剂化结合使用，才能区分我们模型肽的顺式/反式异构体，这表明在转移过程中或处于气相时的任何热激活都会导致异构体混淆。此外，使用18-冠-6的必要性表明，电喷雾电离过程中发生的分子内电荷溶剂化可以克服顺式/反式异构体的同质性。总体而言，结果表明溶液相顺式/反式脯氨酸异构体很脆弱，在电喷雾过程中很容易丢失，需要仔细选择仪器参数并考虑电荷溶剂化以防止顺式/反式混淆。
