Energetics Of Peptide Ion Fragmentation in MALDI/TOF

我们正在使用基质辅助激光解吸电离（MALDI）的肽作为模型系统，研究肽离子碎裂。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。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初步研究的基础上的碎片。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。LeuEnk裂解光谱已被收购在MS和MS-MS操作模式作为激光能量密度的函数开始在电离的开始，并扩展到仪器中可用的最大能量密度。这些光谱揭示了LeuEnk碎裂的几个不同过程。首先，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。我们已经能够将这些快速碎裂，导致亚铵离子，与被广泛接受的是激光脉冲诱导的直接蒸发的分子从样品表面。还有第二组过程发生在激光脉冲之后的最初几百纳秒内。这些过程也表现在MS模式中，最有可能与LeuEnk离子从表面烧蚀的颗粒中解吸有关。这些解吸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;这些碎裂在一系列连续反应中进行，其中酰胺主链键断裂。我们的光谱表明，最初的直接解吸过程达到最大程度，然后不再增加，连续的碎裂反应取代它们的强度。最后，MS-MS模式谱表现出很小的碎片化，最有可能是由于与第二阶段（粒子解吸过程）相关的能量分布的高能部分的耗尽，如上所述。我们同时开发的动力学模型，这些分解使用赖斯-拉姆斯伯格-卡塞尔-马库斯（RRKM）的形式主义气相动力学。除了对碎片化建模之外，计算还将定义肽离子温度的下限。有了这些信息，我们将能够第一次估计激光能量传递到气相离子的分数。此外，我们将有一种手段，而不是纯粹的结晶，选择和优化MALDI矩阵和激光频率。

We 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. We have used the fragmentation of a model peptide, leucine enkephalin, YGGFL, (LeuEnk) over the full range of laser fluence as the basis of the initial studies. 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. LeuEnk fragmentation spectra have been acquired in both MS and MS-MS modes of operation as a function of laser fluence beginning at the onset of ionization and extending to the maximum fluence available in the instrument. These spectra reveal several distinct processes in LeuEnk fragmentation. First, 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. We have been able to associate these rapid fragmentations, leading to immonium ions, with what is widely accepted to be the laser pulse-induced direct vaporization of molecules from the sample surface. There is a second set of process that take place within the first several hundred nanoseconds following the laser pulse. These processes, also manifest in MS mode, are most likely associated with desorption of LeuEnk ions from particles ablated from the surface. These desorbed ions 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. Our spectra show that the initial direct desorption processes reach a maximum extent, and then increase no further, and that the consecutive fragmentation reactions supplant them in intensity. Finally, the MS-MS mode spectra exhibit little fragmentation, most likely due to depletion of the high-energy portions of the energy distributions associated with the second stage, particle desorption processes, described above. We are simultaneously developing a kinetic model for these decompositions using the Rice-Ramsberger-Kassel-Marcus (RRKM) formalism for gas phase kinetics. In addition to modeling fragmentation, the calculations will define a lower limit of the peptide ion temperatures. With this information, we will be able for the first time to estimate the fraction of laser energy delivered to gas phase ions. Furthermore, we will have a means, other than pure empiricism, to select and optimize both MALDI matrix and laser frequency.