Energetics Of Peptide Ion Fragmentation in MALDI/TOF

正在使用基质辅助激光解吸电离（MALDI）的肽作为模型系统，研究肽离子断裂。激光能量密度与肽离子碎裂之间的离子能量学关系。这种类型的研究是优化MALDI TOF/TOF实验的基础，用于肽测序。在这些研究中，我们获得肽片段化光谱，通常5000激光射击，在单分子分解和碰撞诱导解离（CID）模式。我们能够容易地跟踪每个肽分解的两个时间点，即，源内碎裂由激光发射后1微秒内形成的离子组成，而更长的、依赖于质量的碎裂发生在仪器的碰撞室内。到目前为止，我们已经使用了模型肽，亮氨酸脑啡肽，YGGFL，（LeuEnk）在整个范围内的激光能量密度的碎片。虽然不是蛋白质表征中通常遇到的类型的肽，但LeuEnk是一个很好的模型，可以研究激光羽流中的短暂过程，并通过其他质谱方法进行了广泛的研究。光谱作为激光能量密度的函数获得，从电离开始并延伸到仪器中可用的最大能量密度。我们前面已经表明，MS模式光谱显示了一个区域的广泛的碎片发生在什么必须是一个非常短的时间框架后，电离的开始。这些快速碎裂，导致观察到的只有亚铵离子，与低激光羽密度。第二组过程发生在激光脉冲之后的前几百纳秒内，并且发生在高得多的激光羽流密度的范围内。在这些过程中形成的这些离子与激光羽流中存在的高温气体发生大量碰撞，并开始碎裂;这些碎裂在一系列连续反应中进行，其中酰胺主链键断裂。最后，LeuEnk的MS-MS模式光谱表现出很小的碎裂。 我们已经发现，我们的努力，在开发一个动力学模型，这些分解使用赖斯-拉姆斯伯格-卡塞尔-马库斯（RRKM）的气相动力学形式主义的激光羽内的条件有一些不充分的规格。为了解决这一困难，我们进行了所谓的测量？温度计？分子使用尽可能类似于LeuEnk的条件。?温度计？分子是一系列取代的苄基吡啶盐，其具有仅单一断裂反应的重要特征-N-苄基键的断裂。初步结果显示了预期的碎片化，我们目前正试图用理论模型来合理化观测结果。

are using matrix-assisted laser desorption ionization (MALDI) of peptides as a model system to study peptide ion fragmentation. Ion energetics relationships between laser fluence and peptide ion fragmentation. This type of study is fundamental to optimizing MALDI TOF/TOF experiments for the purpose of peptide sequencing. In these studies we obtain peptide fragmentation spectra, typically 5000 laser shots, in both the unimolecular decomposition and collision induced dissociation (CID) modes. We have the ability to easily follow two time points for each peptide decomposition, i.e., the in-source fragmentation consisting of ions formed within 1 usec after the laser firing and the longer, mass dependent fragmentation occurring within the instrument's collision cell. To date we have used the fragmentation of a model peptide, leucine enkephalin, YGGFL, (LeuEnk) over the full range of laser fluence. While not a peptide of the type normally encountered in protein characterizations, LeuEnk is an excellent model to enable studies of short lived processes in the laser plume and been studied extensively by other mass spectrometric approaches. Spectra are acquired as a function of laser fluence beginning at the onset of ionization and extending to the maximum fluence available in the instrument. We have shown earlier that the MS mode spectra show a region of extensive fragmentation occurring in what must be a very short time frame following the onset of ionization. These rapid fragmentations, leading to the observation only of immonium ions, are associated with low laser plume densities. A second set of process takes place within the first several hundred nanoseconds following the laser pulse and occur in the realm of much higher laser plume density. These ions formed in these processes undergo a large number of collisions with the high temperature gases present in the laser plume, and begin to fragment; these fragmentations proceed in a series of consecutive reactions in which the amide backbone bonds are ruptured. Finally, the MS-MS mode spectra of LeuEnk exhibit little fragmentation. We have found that our efforts in developing a kinetic model for these decompositions using the Rice-Ramsberger-Kassel-Marcus (RRKM) formalism for gas phase kinetics have somewhat of an inadequate specification of the conditions within the laser plume. In order to address this difficulties, we have undertaken the measurement of so called ?thermometer? molecules using conditions that are as similar as possible to those used for the LeuEnk. ?Thermometer? molecules are a series of substituted benzyl pyridinium salts that have the important characteristic of only a single fragmentation reaction - the breaking of the N-benzyl bond. The initial results show the expected fragmentation and we are currently attempting to rationalize the observations with the theoretical model.