Warnke Stephan, von Helden Gert, Pagel Kevin
Fritz Haber Institute of the Max Planck Society, Berlin, Germany.
Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany.
Proteomics. 2015 Aug;15(16):2804-12. doi: 10.1002/pmic.201400480. Epub 2015 Apr 27.
The top-down approach in protein sequencing requires simple methods in which the analyte can be readily dissociated at every position along the backbone. In this context, ultraviolet photodissociation (UVPD) recently emerged as a promising tool because, in contrast to slow heating techniques such as CID, the absorption of UV light is followed by a rather statistically distributed cleavage of backbone bonds. As a result, nearly complete sequence coverage can be obtained. It is well known, however, that gas-phase proteins can adopt a variety of different, sometimes coexisting conformations and the influence of this structural diversity on the UVPD fragmentation behavior is not clear. Using ion mobility-UVPD-MS, we recently showed that UVPD is sensitive to the higher order structure of gas-phase proteins. In particular, the cis/trans isomerization of certain proline peptide bonds was shown to significantly influence the UVPD fragmentation pattern of two extended conformers of 11(+) ubiquitin. Building on these results, we here provide conformer-selective UVPD data for 7(+) ubiquitin ions, which are known to be present in a much more diverse and wider ensemble of different structures, ranging from very compact to highly extended species. Our data show that certain conformers fall into groups with similar UVPD fragmentation pattern. Surprisingly, however, the conformers within each group can differ tremendously in their collision cross-section. This indicates that the multiple coexisting conformations typically observed for 7(+) ubiquitin are caused by a few, not easily interconvertible, subpopulations.
蛋白质测序中的自上而下方法需要简单的方法,使分析物能够在沿着主链的每个位置容易地解离。在这种情况下,紫外光解离(UVPD)最近成为一种很有前景的工具,因为与诸如碰撞诱导解离(CID)等缓慢加热技术不同,紫外光的吸收之后是主链键的相当随机分布的断裂。结果,可以获得几乎完整的序列覆盖。然而,众所周知,气相蛋白质可以采取多种不同的、有时共存的构象,而这种结构多样性对UVPD碎片化行为的影响尚不清楚。利用离子淌度-UVPD-质谱,我们最近表明UVPD对气相蛋白质的高级结构敏感。特别是,某些脯氨酸肽键的顺/反异构化被证明会显著影响11(+)泛素的两种伸展构象的UVPD碎片化模式。基于这些结果,我们在此提供了7(+)泛素离子的构象选择性UVPD数据,已知7(+)泛素离子存在于更加多样和广泛的不同结构集合中,范围从非常紧凑到高度伸展的物种。我们的数据表明,某些构象可分为具有相似UVPD碎片化模式的组。然而,令人惊讶的是,每组内的构象在其碰撞截面方面可能有很大差异。这表明通常在7(+)泛素中观察到的多种共存构象是由少数几个不易相互转化的亚群引起的。