Nanohydrophobic Interaction Chromatography Coupled to Ultraviolet Photodissociation Mass Spectrometry for the Analysis of Intact Proteins in Low Charge States
Nanohydrophobic Interaction Chromatography Coupled to Ultraviolet Photodissociation Mass Spectrometry for the Analysis of Intact Proteins in Low Charge States
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DOI：
10.1021/acs.jproteome.2c00450
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发表时间：
2022-08-31
影响因子：
4.4
通讯作者：
Brodbelt, Jennifer S.
中科院分区：
文献类型：
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作者：
Blevins, Molly S.;Juetten, Kyle J.;Brodbelt, Jennifer S.
蛋白质型与生物表型之间的直接相关性需要探索更适合蛋白质型检测和表征的基于质谱（MS）的方法。在这里，我们将纳米疏水相互作用色谱 (nano-HIC) 与紫外光解离 MS (UVPD-MS) 结合起来，以分离和表征完整的蛋白质和蛋白质形式。对于多种蛋白质，该方法可以获得高线性度、灵敏度和序列覆盖率。对纳米 HIC 过程中完整蛋白质碰撞截面的研究表明，其在低电荷态下具有半折叠构象，与传统的完全变性反相分离相比，能够实现不同维度的分离。该方法已针对来自大肠杆菌核糖体的完整蛋白质混合物进行了验证；多种修饰和未修饰的蛋白质形式获得了高序列覆盖率。
The direct correlation between proteoforms and biological phenotype necessitates the exploration of mass spectrometry (MS)-based methods more suitable for proteoform detection and characterization. Here, we couple nano-hydrophobic interaction chromatography (nano-HIC) to ultraviolet photodissociation MS (UVPD-MS) for separation and characterization of intact proteins and proteoforms. High linearity, sensitivity, and sequence coverage are obtained with this method for a variety of proteins. Investigation of collisional cross sections of intact proteins during nano-HIC indicates semifolded conformations in low charge states, enabling a different dimension of separation in comparison to traditional, fully denaturing reversed-phase separations. This method is demonstrated for a mixture of intact proteins from Escherichia coli ribosomes; high sequence coverage is obtained for a variety of modified and unmodified proteoforms.