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，源内碎裂包括在激光器发射后1微秒内形成的离子，而更长的、依赖于质量的碎裂发生在仪器碰撞室内。到目前为止，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，并通过其他质谱方法进行了广泛的研究。光谱作为激光能量密度的函数获得，从电离开始并延伸到仪器中可用的最大能量密度。我们前面已经表明，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。这些快速碎裂，导致观察到的只有亚铵离子，与低激光羽密度。第二组过程发生在激光脉冲之后的前几百纳秒内，并且发生在高得多的激光羽流密度的范围内。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。最后，LeuEnk的MS-MS模式光谱表现出很小的碎裂。 我们最初的研究是使用5纳秒持续时间的激光脉冲长度进行的。如上所述，这些研究表明，碎裂程度随激光能量密度而变化。也就是说，片段化的总程度随着能量密度的增加而增加，并且片段以与肽中氨基酸序列一致的顺序出现。从肽的氨基和羧基末端发生连续断裂。在最近的研究中，我们使用0.6纳秒持续时间的激光脉冲长度研究了这些相同的现象，并确定了三种不同的常见MALDI基质中的每一种的碎片：α氰基-4-羟基肉桂酸（ACHA），3，5-二甲氧基-4-羟基肉桂酸（芥子酸，SA）和2，5-二羟基苯甲酸（DHB）。在后两种基质材料中，观察到非常少的碎片，正如从被广泛认为比ACHA更冷的基质中所预期的那样，但DHB始终显示出比SA更广泛的碎片。然而，明显更令人惊讶的是，ACHA中广泛碎裂的开始发生在比使用5纳秒脉冲长度激光观察到的低约50%的激光能量密度下。这些意见意味着，在固体表面上的能量浓度进行MALDI，在光子/单位面积/单位时间，有一个显着的影响的程度上观察到的光谱的碎片。

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 instruments 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. Our initial studies were conducted using a laser pulse length of 5 nsec duration. as summarized above, these studies showed that the extent of fragmentation varied with laser fluence. That is, the total extent of fragmentation increased as fluence increased and the fragments appeared in an order consistent with the sequence of amino acids in the peptide. The sequential fragmentation occurred from both the amino and carboxy terminals of the peptide. In more recent studies, we have investigated these same phenomena using a laser pulse length of 0.6 nsec duration, and have determined the fragmentation in each of three different common MALDI matrices: alpha cyano-4-hydroxy cinamic acid (ACHA), 3,5-dimethoxy-4-hydroxy cinamic acid (Sinapinic acid, SA) and 2,5-dihydroxy benzoic acid (DHB). In both of the latter matrix materials, very little fragmentation was observed, as might be expected from matrices that are widely considered to be cooler than ACHA, but the DHB consistently showed more extensive fragments than the SA. What was appreciably more surprising however is that the onset of extensive fragmentation in ACHA occurred at laser fluences about 50% lower than than observed using the 5 nsec pulse length laser. These observations imply that the concentration of energy on the solid surface undergoing MALDI, in terms of photons/unit area/unit time, has a dominant influence on the extent the fragmentation that is observed in spectra.