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。到目前为止，我们已经使用了一个模型肽，亮氨酸脑啡肽，YGGFL，(LeuEnk)在整个激光通量范围内的碎裂。虽然LeuEnk不是蛋白质表征中通常遇到的那种类型的肽，但它是一个出色的模型，能够研究激光羽流中的短暂过程，并通过其他质谱学方法进行了广泛的研究。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。我们早些时候已经表明，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能观察到免疫离子，这与低激光羽流密度有关。第二组过程在激光脉冲后的最初几百纳秒内发生，并且发生在激光羽流密度高得多的领域。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。最后，LeuEnk的MS-MS模式谱几乎没有碎裂。 我们最初的研究是使用持续时间为5纳秒的激光脉冲进行的。如上所述，这些研究表明，破碎的程度随着激光通量的不同而不同。也就是说，总的碎裂程度随着剂量的增加而增加，并且片段的出现顺序与肽中的氨基酸序列一致。该肽的氨基末端和羧基末端都发生了顺序断裂。在最近的研究中，我们用0.6毫微秒的激光脉冲研究了同样的现象，并测定了三种不同的MALDI基质：α氰基-4-羟基肉桂酸(ACHA)、3，5-二甲氧基-4-羟基肉桂酸(SA)和2，5-二羟基苯甲酸(DHB)中的裂解。在后两种基质材料中，几乎没有观察到碎裂，这可以从被广泛认为比ACHA更冷的基质中观察到，但DHB始终显示出比SA更广泛的碎片。然而，更令人惊讶的是，ACHA的广泛破碎发生在比使用5纳秒脉冲长度激光观察到的激光通量低约50%的情况下。这些观测表明，在经历MALDI的固体表面上的能量集中，以光子/单位面积/单位时间为单位，对在光谱中观察到的碎裂程度具有主导影响。

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.