Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
Methods Mol Biol. 2022;2393:207-224. doi: 10.1007/978-1-0716-1803-5_11.
Recent advances in nanoscale separations and high-resolution mass spectrometry permit highly sensitive and accurate analyses of complex protein mixtures. Here, we describe improved methods for nanoscale multidimensional chromatography coupled to targeted mass spectrometry (tMS) to achieve ultrasensitive quantification of peptides in complex proteomes. The presented chromatographic system consists of capillary strong-cation exchange (SCX) chromatography column, from which peptides are eluted directly onto high-resolution reversed-phase (RP) analytical columns and nanoelectrospray ion source. SCX prefractionation is used to separate phosphorylated peptides, permitting their ultrasensitive quantification. Resolution and robustness of this chromatographic system, together with the orthogonality of SCX and RP separations, permit scheduling of large panels of targeted MS assays. This design also enables seamless scaling to three-dimensional separations, thereby enabling large-scale, ultrasensitive quantitative proteomics.
近年来，纳米尺度分离和高分辨率质谱技术的进展使得对复杂蛋白质混合物进行高灵敏度和高准确度分析成为可能。在这里，我们描述了改进的纳米多维色谱与靶向质谱（tMS）联用方法，以实现复杂蛋白质组中肽的超灵敏定量。所提出的色谱系统由毛细管强阳离子交换（SCX）色谱柱组成，其中肽直接从 SCX 色谱柱洗脱到高分辨率反相（RP）分析柱和纳喷雾离子源。SCX 预分级用于分离磷酸化肽，从而能够对其进行超灵敏定量。该色谱系统的分辨率和稳健性，以及 SCX 和 RP 分离的正交性，允许对大量靶向 MS 测定进行计划。这种设计还能够无缝扩展到三维分离，从而实现大规模、超灵敏的定量蛋白质组学。
