Blevins Molly S, Juetten Kyle J, James Virginia K, Butalewicz Jamie P, Escobar Edwin E, Lanzillotti Michael B, Sanders James D, Fort Kyle L, Brodbelt Jennifer S
Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States.
Thermo Fisher Scientific, Bremen 28199, Germany.
J Proteome Res. 2022 Oct 7;21(10):2493-2503. doi: 10.1021/acs.jproteome.2c00450. Epub 2022 Aug 31.
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 ribosomes; high sequence coverage is obtained for a variety of modified and unmodified proteoforms.
蛋白质变体与生物学表型之间的直接关联使得有必要探索更适合蛋白质变体检测和表征的基于质谱（MS）的方法。在此，我们将纳米疏水相互作用色谱（nano-HIC）与紫外光解离质谱（UVPD-MS）联用，用于完整蛋白质和蛋白质变体的分离与表征。该方法对多种蛋白质具有高线性、高灵敏度和高序列覆盖率。对纳米疏水相互作用色谱过程中完整蛋白质的碰撞截面进行研究表明，低电荷状态下存在半折叠构象，与传统的完全变性反相分离相比，这实现了不同维度的分离。该方法已用于核糖体完整蛋白质混合物的分析；对于多种修饰和未修饰的蛋白质变体均获得了高序列覆盖率。
