Electrospray ionization mass spectrometry as a tool to analyze hydrogen/deuterium exchange kinetics of transmembrane peptides in lipid bilayers
Electrospray ionization mass spectrometry as a tool to analyze hydrogen/deuterium exchange kinetics of transmembrane peptides in lipid bilayers
复制标题
DOI：
10.1073/pnas.050444797
复制
发表时间：
2000-03-28
影响因子：
11.1
通讯作者：
Killian, JA
中科院分区：
文献类型：
--
作者：
Demmers, JAA;Haverkamp, J;Killian, JA
描述了一种结合氢/氘（H/D）交换和纳米电喷雾电离质谱来研究跨膜肽在磷脂双层中的精确定位的方法。该方法通过使用由设计的α-螺旋跨膜肽[乙酰基GW（2）（LA）（5）W（2）乙醇胺（WALP 16）和乙酰基（CA）（3）W-2（LA）（5）W-2（AG）（3）乙醇胺（WALP 16（+10））]组成的模型系统进行测试，所述模型系统并入1，2-二肉豆蔻酰-sn-甘油-3-磷酸胆碱的大单层囊泡中。Bath肽由交替的亮氨酸/丙氨酸疏水核心序列组成，其侧翼为作为界面锚残基的色氨酸残基。在WALP 16（+10）的情况下，该序列在两端通过延伸到双层周围的水相中的5-aa甘氨酸/丙氨酸尾延伸，在缓冲的氧化氘中稀释囊泡后及时监测这些肽中不稳定氢的H/D交换。发现肽可以通过将蛋白脂质体悬浮液直接引入质谱仪中来测量。观察到几种不同的H/D交换率（对应于半衰期值从小于或等于2到大约2 x 10（4）min不等）。对于WALP 16和WALP 16（+10），接近膜/水界面的残基具有中间交换速率，而膜包埋的疏水核心具有缓慢交换速率。这些分配证实了碰撞诱导解离串联质谱实验的结果，这使得分析交换的个别肽酰胺键。这种蛋白脂质体纳米电喷雾电离质谱技术被证明是一种极其敏感且强大的工具，可以揭示肽-膜相互作用的位点特异性信息。
A method is described to study the precise positioning of transmembrane peptides in a phospholipid bilayer combining hydrogen/deuterium (H/D) exchange and nanoelectrospray ionization mass spectrometry. The method was tested by using model systems consisting of designed alpha-helical transmembrane peptides [acetylGW(2)(LA)(5)W(2)Aethanolamine (WALP16) and acetyl(CA)(3)W-2(LA)(5)W-2(AG)(3)ethanolamine (WALP16(+10))] incorporated in large unilamellar Vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Bath peptides consist of an alternating leucine/ alanine hydrophobic core sequence flanked by tryptophan residues as interfacial anchor residues, In the case of WALP16(+10), this sequence is extended at both ends by 5-aa glycine/alanine tails extending into the aqueous phase surrounding the bilayer, H/D exchange of labile hydrogens in these peptides was monitored in time after dilution of the vesicles in buffered deuterium oxide. It was found that the peptides can be measured by direct introduction of the proteoliposome suspension into the mass spectrometer. Several distinct H/D exchange rates were observed (corresponding to half-life values varying from less than or equal to 2 to approximate to 2 x 10(4) min). Fast exchange rates were assigned to the water-exposed tails of WALP16(+10), For both WALP16 and WALP16(+10), intermediate exchange rates were assigned to the residues close to the membrane/water interface, and the slow exchange rates to the membrane-embedded hydrophobic core. These assignments were confirmed by results from collision-induced dissociation tandem mass spectrometry experiments, which allowed analysis of exchange of individual peptide amide linkages. This proteoliposome nanoelectrospray ionization mass spectrometry technique is shown to be an extremely sensitive and powerful tool for revealing site-specific information on peptide-membrane interactions.