Energetics Of Peptide Ion Fragmentation in MALDI/TOF

利用多肽的基质辅助激光解吸电离（MALDI）作为模型体系来研究多肽离子的断裂。激光能量流与肽离子碎裂的离子能量学关系。这种类型的研究是优化MALDI TOF/TOF实验以进行肽测序的基础。在这些研究中，我们在单分子分解和碰撞诱导解离（CID）模式下获得了肽碎片光谱，通常是5000次激光射击。我们能够很容易地跟踪每个肽分解的两个时间点，即激光发射后1秒内形成的由离子组成的源内碎片和仪器碰撞单元内发生的更长时间的质量依赖碎片。到目前为止，我们已经使用了一种模型肽的碎片，亮氨酸脑啡肽，YGGFL, （LeuEnk）在整个激光影响范围内。虽然不是蛋白质表征中通常遇到的肽类型，但LeuEnk是一个很好的模型，可以研究激光羽流中的短寿命过程，并通过其他质谱方法进行了广泛的研究。光谱是作为激光能量的函数获得的，从电离开始一直延伸到仪器中可用的最大能量。我们先前已经表明，质谱模式光谱显示在电离开始后的很短时间内发生了广泛的碎片化区域。这些快速的碎片，导致只能观察到铵离子，与低激光羽流密度有关。第二组过程发生在激光脉冲后的最初几百纳秒内，发生在激光羽流密度高得多的区域。在这些过程中形成的离子与激光羽流中的高温气体发生大量碰撞，并开始碎裂；这些断裂在酰胺主键断裂的一系列连续反应中进行。最后，LeuEnk的MS-MS模式谱呈现较小的碎片化。

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.