Photodissociation and Dissociative Photoionization Mass Spectrometry of Proteins and Noncovalent Protein-Ligand Complexes
Photodissociation and Dissociative Photoionization Mass Spectrometry of Proteins and Noncovalent Protein-Ligand Complexes
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DOI：
10.1002/anie.201304046
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发表时间：
2013-08-05
影响因子：
16.6
通讯作者：
Giuliani, Alexandre
中科院分区：
文献类型：
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作者：
Canon, Francis;Milosavljevic, Aleksandar R.;Giuliani, Alexandre
串联质谱 (MS2) 是结构分析和生物聚合物测序中广泛使用的方法。 [1]在 MS2 中，目标离子被分离、激活并解离。经过分析后，生成的碎片提供了母离子的结构信息。[2]在不同的激活方法中，低能碰撞诱导解离（CID）是应用最广泛的，它依赖于离子通过与中性气体的多次非弹性低能碰撞缓慢加热。 [3]另一组激活技术，称为 EXD 方法（电子捕获解离 (ECD) 或电子转移解离 (ETD)），涉及多质子化离子与电子的解离重组反应。 [4] EXD 技术集保留了不稳定的键，这使得它们特别适合定位蛋白质骨架上的翻译后修饰 (PTM) 和非共价结合位点。[3, 5] 增加离子内能的另一种方法是吸收高能光子。自 Williams 和 McLafferty [6] 的开创性工作以来，涉及基于紫外 (UV) 激光的电喷雾离子光解离 (PD) 的方法引起了越来越多的兴趣，并且最近在蛋白质组学分析中发现了有前景的应用。 [7]事实上，紫外线激活产生的片段与其他方法产生的片段互补，并提供肽序列的高覆盖率，这几乎是可以从实验中确定的完整序列。在使用的不同波长中，193 nm (6.2 eV) 和 157 nm (7.8 eV) 的真空紫外 (VUV) 光子已被证明具有最高的使用潜力。 [7, 8] 然而，人们对 8 eV 以上引起的碎片知之甚少。这个问题与激光器传递的光子能量的限制有关。令人惊讶的是，同步辐射 (SR) 是 VUV 中广泛可调的光子源，但直到最近才被用于离子激活。初步工作表明有可能带来小肽的序列信息。 [9]在此，我们报告了 SR 作为 MS2 中用于结构分析的可靠激活方法的潜力。在蛋白质序列覆盖方面，将宽能量范围内的光子激活性能与 CID 和 ECD 的性能进行了比较。更有趣的是，我们研究了这种新方法保留非共价相互作用并允许识别蛋白质上配体结合位点的能力，并将其结果与 ECD 获得的结果进行比较。这项研究是针对一种名为 IB5 的人类本质无序蛋白质 (IDP) 进行的。 IB5 和其他富含脯氨酸的碱性蛋白 (PRP) 唯一已知的功能是结合和清除单宁，从而构成对抗单宁抗营养作用的第一道防线。这些相互作用也被认为在涩味感中发挥着作用，[10]这是食品关于消费者可接受性的最重要的感官特性之一。迄今为止，IB5 上的单宁结合位点无法通过经典结构方法（例如 X 射线衍射或 NMR 光谱）精确确定，因为缺乏用于衍射的晶体，并且由于该蛋白质的多个重复序列而导致光谱解释困难。相比之下，本文提出的基于 SR 的串联质谱激活方法首次实现了单宁原花青素 B2 3оOG（(À)-表儿茶素 (4β-8)-(À)-表儿茶素 3-O-没食子酸酯）在 IB5 上的结合位点的明确测定。
Tandem mass spectrometry (MS2) is a widely used method in structural analysis and biopolymer sequencing.[1] In MS2, an ion of interest is isolated, activated, and brought to dissociation. After analysis, the generated fragments provide structural information on the precursor ion.[2] Among the different activation methods, low-energy collision induced dissociation (CID), which relies on slow heating of ions through multiple inelastic low-energy collisions with a neutral gas, is the most widely used.[3] Another set of activation techniques, referred to as the EXD methods (electron capture dissociation (ECD) or electron-transfer dissociation (ETD)), involves the dissociative recombination reaction of multiply protonated ions with electrons.[4] The EXD set of techniques preserve labile bonds, which makes them particularly suited to localize post-translational modifications (PTM) and noncovalent binding sites on protein backbones.[3, 5] Another way to increase the internal energy of an ion is by absorption of energetic photons. Methods involving ultraviolet (UV) laserbased photodissociation (PD) of electrosprayed ions are attracting a growing interest since the pioneering work of Williams and McLafferty [6] and have recently found promising applications in proteomics analysis.[7] Indeed, UV activation produces fragments complementary to those generated by other methods and provides high coverage in peptide sequence, that is almost the complete sequence can be determined from the experiments. Among the different wavelengths used, vacuum ultraviolet (VUV) photons of 193 nm (6.2 eV) and 157 nm (7.8 eV) have demonstrated the highest potential to be of use.[7, 8] However, very little is known about fragmentations induced above 8 eV. This problem pertains to the limitations in terms of photon energy delivered by lasers. Surprisingly, synchrotron radiation (SR), which is a widely tunable photon source in the VUVs, has been used only very recently for ion activation. Preliminary work has indicated some potential to bring sequence information on small peptides.[9] Herein, we report on the potential of SR as a credible activation method in MS2 for structural analysis. The performances of photon activation over a wide energy range are compared to those of CID and ECD in terms of protein sequence coverage. More interestingly, the ability of this new method to preserve noncovalent interactions and to permit identification of the binding sites of a ligand on a protein is investigated and its outcomes are compared to those obtained by ECD.This study has been performed on a human intrinsically disordered protein (IDP), named IB5. The only known function of IB5 and other basic proline-rich proteins (PRPs) is to bind and scavenge tannins, and thus constitute a first-line of defense against tannin anti-nutritional effects. These interactions are also thought to play a role in the sensation of astringency,[10] which is one of the most important organoleptic properties of food regarding consumer acceptability. Hitherto, the tannin binding sites on IB5 could not be precisely determined by classical structural approaches, such as X-rays diffraction or NMR spectroscopy, because of the lack of crystals for diffraction and of difficulties in spectra interpretation for spectroscopy because of the multiple repeated sequences of this protein. In contrast, the SR-based tandem mass spectrometry activation method presented herein has achieved, for the first time, unambiguous determination of the binding site of the tannin procyanidin B2 3оOG ((À)-epicatechin (4β-8)-(À)-epicatechin 3-O-gallate) on IB5.