Energetics Of Peptide Ion Fragmentation in MALDI/TOF

我们将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。我们使用了一种模型肽，leucine enkephalin， YGGFL, （LeuEnk）在全范围激光影响下的碎片作为初步研究的基础。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。在质谱和质谱两种操作模式下，获得了从电离开始到仪器中可用的最大激光通量的函数，即LeuEnk碎片谱。这些光谱揭示了LeuEnk碎裂的几个不同过程。首先，质谱模式显示在电离开始后很短的时间内出现了广泛的碎片。我们已经能够将这些导致铵离子的快速碎片，与被广泛接受的激光脉冲诱导的样品表面分子的直接汽化联系起来。在激光脉冲后的最初几百纳秒内还会发生第二套过程。这些过程，也在质谱模式中表现出来，很可能与从表面烧蚀的粒子中脱附LeuEnk离子有关。这些被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；这些断裂在酰胺主键断裂的一系列连续反应中进行。我们的光谱表明，初始直接脱附过程达到最大程度，然后不再增加，连续的裂解反应在强度上取代了它们。最后，MS-MS模式谱显示出很少的碎片化，这很可能是由于与上述第二阶段（粒子脱附过程）相关的能量分布的高能部分耗尽。同时，我们正在使用Rice-Ramsberger-Kassel-Marcus （RRKM）气相动力学形式来开发这些分解的动力学模型。除了模拟碎片，计算还将定义肽离子温度的下限。有了这些信息，我们将能够首次估计激光能量传递给气相离子的比例。此外，我们将有一种方法，而不是纯粹的经验主义，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.