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)多肽作为研究多肽离子碎裂的模型系统。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。我们使用了一个模型肽，亮氨酸脑啡肽，YGGFL，(LeuEnk)在整个激光通量范围内的碎裂作为初步研究的基础。虽然LeuEnk不是通常在蛋白质表征中遇到的那种类型的肽，但它是一个出色的模型，可以研究激光羽流中的短暂过程。在MS和MS-MS中使用三种不同的常见矩阵获得了LeuEnk碎裂光谱，使用的激光脉冲长度为600PSEC，而以前使用的是5毫微秒。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。用α-氰基-羟基肉桂酸(ACHA)获得的光谱揭示了LeuEnk碎裂的几个不同过程。首先，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观察到免疫离子，与激光脉冲诱导样品表面的分子直接汽化有关。第二组过程发生在激光脉冲后的最初几百纳秒内。这些过程也表现在MS模式下，很可能与LeuEnk离子从颗粒上解吸有关，这些颗粒主要由基质组成，从样品表面烧蚀。这些解吸的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。我们的光谱表明，最初的直接脱附过程在约占总激光通量的50%时达到最大值，然后不再进一步增加；在这一点上，连续的碎裂反应在强度上取代了它们。最后，MS-MS模式光谱几乎没有碎裂，这很可能是由于与上述第二阶段，即粒子解吸过程相关联的能量分布的高能部分耗尽所致。使用2，5-二羟基苯甲酸(DHB)或二甲氧基羟基肉桂酸(芥子酸，SA)可以得到比ACHA观察到的光谱碎片少得多的光谱，而质子化LeuEnk分子本身的起始通量要高得多。与ACHA观察到的大量免疫离子的产生相反，使用SA根本看不到免疫离子的产生，使用DHB观察到的水平约为ACHA的30%。与ACHA相比，SA和DHB中较低水平的免疫离子也与较低水平的脊椎碎裂有关。最后，用600PSEC的激光脉冲长度与使用5nSec脉冲长度的早期研究进行了比较。这项研究表明，600PSEC脉冲长度在低得多的注量水平下引起电离开始，并且与5纳秒脉冲长度相比，与高水平的免疫离子形成有关。这些观察结果几乎可以肯定是上述第一种机制占主导地位的结果，即从样品表面直接解吸。

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.