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MS和MS-MS中，使用三种不同的常见矩阵，使用600 psec的激光脉冲长度获得了LeuEnk碎片谱，而之前使用的激光脉冲长度为5 nsec。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。利用a-氰基羟基肉桂酸（ACHA）获得的光谱揭示了LeuEnk断裂的几个不同过程。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。这些快速的碎片，导致只观察到铵离子，与激光脉冲诱导的样品表面分子的直接汽化有关。第二套过程在激光脉冲后的几百纳秒内发生。这些过程也在质谱模式中表现出来，很可能与从样品表面烧蚀的主要由基质组成的颗粒中脱附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，初始直接解吸过程在激光总通量的50%左右达到最大值，然后不再增加；在这一点上，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。使用2,5-二羟基苯甲酸（DHB）或二甲氧基羟基肉桂酸（sinapinic acid， SA）可导致比使用ACHA观察到的光谱更少的碎片化，并且对质子化的LeuEnk分子本身的起始具有更高的影响。与用ACHA观察到的大量产生铵离子相反，用SA观察到的完全没有，用DHB观察到的水平大约是用ACHA观察到的30%。与ACHA相比，SA和DHB中较低水平的铵离子也与较低水平的骨干断裂有关。最后，比较了600 psec激光脉冲长度与早期5nsec激光脉冲长度对ACHA破碎的影响。这项研究表明，600秒脉冲长度比5秒脉冲长度在更低的影响水平下引起电离，并且与更高水平的铵离子形成有关。这些观察结果几乎肯定是上述第一种机制占主导地位的结果，即样品表面的直接解吸。

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 using three different common matrices using a laser pulse length of 600 psec, compared to 5 nsec used previously. 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. The spectra obtained using a-cyano-hydroxycinnamic acid (ACHA) 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. These rapid fragmentations, leading to the observation only of immonium ions, are associated with the laser pulse-induced direct vaporization of molecules from the sample surface. A second set of process takes 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, consisting principally of matrix, ablated from the sample 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 at about 50% of the total laser fluence, and then increase no further; at that point, 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. Use of either 2,5-dihydroxy benzoic acid (DHB) or di-methoxy-hydroxy cinnamic acid (sinapinic acid, SA) leads to spectra with much less fragmentation than observed with ACHA coupled with a much higher fluence for the onset of the protonated LeuEnk molecule itself. in contrast to the extensie production of immonium ions observed with ACHA, none at all are seen with the use of SA and the levels observed using DHB are about 30% of those seen in ACHA. These lower levels of immonium ions in SA and DHB are also associated with much lower levels of backbone fragmentation compared to ACHA. Finally, a comparison of ACHA fragmentation was made using laser pulse length of 600 psec with the earlier studies employing 5 nsec pulse lengths. This study showed that the 600 psec pulse length gives rise to the onset of ionization at much lower levels of fluence and is associated with much higher levels of immonium ion formation than does the 5 nsec puls length. These observations are almost certainly the result of the dominance of the first mechanism described above, the direct desorption from the sample surface.