Energetics Of Peptide Ion Fragmentation in MALDI/TOF

正在使用基质辅助激光解吸电离(MALDI)多肽作为模型系统来研究多肽离子碎裂。激光通量与多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实验以实现肽序列测定的基础。在这些研究中，我们获得了单分子分解和碰撞诱导解离(CID)模式下的多肽碎裂光谱，通常为5000次激光。我们能够轻松跟踪每个肽分解的两个时间点，即由激光激发后1微秒内形成的离子组成的源内碎裂和仪器碰撞单元内发生的更长的、质量相关的碎裂。到目前为止，我们已经使用了一个模型肽，亮氨酸脑啡肽，YGGFL，(LeuEnk)在整个激光通量范围内的碎裂。虽然LeuEnk不是蛋白质表征中通常遇到的那种类型的肽，但它是一个出色的模型，能够研究激光羽流中的短暂过程，并通过其他质谱学方法进行了广泛的研究。光谱是作为激光通量的函数获得的，从电离开始到仪器中可用的最大通量为止。我们早些时候已经表明，MS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裂区域。这些快速碎裂导致只能观察到免疫离子，这与低激光羽流密度有关。第二组过程在激光脉冲后的最初几百纳秒内发生，并且发生在激光羽流密度高得多的领域。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经历了大量的碰撞，并开始碎裂；这些碎裂在一系列连续的反应中进行，在这些反应中，酰胺主键断裂。最后，LeuEnk的MS-MS模式谱几乎没有碎裂。 我们发现，我们使用气相动力学的莱斯-拉姆斯伯格-卡塞尔-马库斯(RRKM)公式来开发这些分解的动力学模型的努力，在某种程度上没有充分说明激光羽流中的条件。为了解决这一困难，我们进行了所谓的温度计的测量。分子使用的条件与LeuEnk使用的条件尽可能相似。体温计？分子是一系列取代的苄基吡啶盐，其重要特征是只有一次裂解反应--N-苄基键的断裂。初步结果显示了预期的碎裂，我们目前正试图用理论模型对观察结果进行合理化。

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.