Abstract：
A recently developed proteolytic reactor, designed for protein structural investigation, was coupled to ion mobility mass spectrometry to monitor collisional cross section (CCS) evolution of model proteins undergoing trypsin-mediated mono enzymatic digestion. As peptides are released during digestion, the CCS of the remaining protein structure may deviate from the classical 2/3 power of the CCS-mass relationship for spherical structures. The classical relationship between CCS and mass (CCS = A × M2/3) for spherical structures, assuming a globular shape in the gas phase, may deviate as stabilizing elements are lost during digestion. In addition, collision-induced unfolding (CIU) experiments on partially digested proteins provided insights into the CCS resilience in the gas phase to ion activation, potentially due to the presence of stabilizing elements. The study initially investigated a model peptide ModBea (3 kDa), assessing the impact of disulfide bridges on CCS resilience in both reduced and oxidized forms. Subsequently, β-lactoglobulin (2 disulfide bridges), calmodulin (Ca2+ coordination cation), and cytochrome c (heme) were selected to investigate the influence of common structuring elements on CCS resilience. CIU experiments probed the unfolding process, evaluating the effect of losing specific peptides on the energy landscapes of partially digested proteins. Comparisons of the TWCCSN2→He to trend curves describing the CCS/mass relationship revealed that proteins with structure-stabilizing elements consistently exhibit TWCCSN2→He and greater resilience toward CIU compared to proteins lacking these elements. The integration of online digestion, ion mobility, and CIU provides a valuable tool for identifying structuring elements in biopolymers in the gas phase.
摘要：
最近开发的蛋白水解反应器,专为蛋白质结构研究而设计，与离子迁移质谱耦合，以监测经历胰蛋白酶介导的单酶消化的模型蛋白质的碰撞横截面（CCS）进化。由于肽在消化过程中释放，其余蛋白质结构的CCS可能偏离球形结构的CCS-质量关系的经典2/3幂。对于球形结构，CCS与质量之间的经典关系（CCS=A×M2/3），假设气相呈球形，可能会偏离稳定元素在消化过程中丢失。此外,碰撞诱导的去折叠（CIU）对部分消化蛋白质的实验提供了对气相中CCS对离子活化的弹性的见解，可能是由于稳定元素的存在。该研究最初研究了模型肽ModBea（3kDa），评估二硫键对还原和氧化形式的CCS弹性的影响。随后，β-乳球蛋白（2二硫键），钙调蛋白（Ca2+配位阳离子），选择细胞色素c（血红素）来研究常见结构元素对CCS弹性的影响。CIU实验探索了展开过程，评估失去特定肽对部分消化蛋白质的能量景观的影响。TWCCSN2→He与描述CCS/质量关系的趋势曲线的比较表明，与缺乏这些元素的蛋白质相比，具有结构稳定元素的蛋白质始终表现出TWCCSN2→He和更大的弹性。在线消化的整合，离子迁移率，和CIU为识别气相生物聚合物中的结构元素提供了有价值的工具。
