Wu Si, Tolić Nikola, Tian Zhixin, Robinson Errol W, Paša-Tolić Ljiljana
Pacific Northwest National Laboratory, Richland, WA, USA.
Methods Mol Biol. 2011;694:291-304. doi: 10.1007/978-1-60761-977-2_18.
Bottom-up and top-down strategies are two commonly used methods for mass spectrometry (MS) based protein identification; each method has its own advantages and disadvantages. In this chapter, we describe an integrated top-down and bottom-up approach facilitated by concurrent liquid chromatography-mass spectrometry (LC-MS) analysis and fraction collection for comprehensive high-throughput intact protein profiling. The approach employs a high resolution reversed phase (RP) LC separation coupled with LC eluent fraction collection and concurrent on-line MS with a high field (12 T) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer. Protein elusion profiles and tentative modified protein identification are made using detected intact protein mass in conjunction with bottom-up protein identifications from the enzymatic digestion and analysis of corresponding LC fractions. Specific proteins of biological interest are incorporated into a target ion list for subsequent off-line gas-phase fragmentation that uses an aliquot of the original collected LC fraction, an aliquot of which was also used for bottom-up analysis.
自下而上和自上而下策略是基于质谱（MS）的蛋白质鉴定中两种常用的方法；每种方法都有其自身的优缺点。在本章中，我们描述了一种通过同时进行液相色谱-质谱（LC-MS）分析和馏分收集来促进的综合自上而下和自下而上的方法，用于全面的高通量完整蛋白质谱分析。该方法采用高分辨率反相（RP）LC分离，结合LC洗脱液馏分收集以及与高场（12 T）傅里叶变换离子回旋共振（FTICR）质谱仪同时进行的在线MS。使用检测到的完整蛋白质质量结合来自相应LC馏分的酶促消化和分析的自下而上蛋白质鉴定，来生成蛋白质洗脱谱和初步的修饰蛋白质鉴定。将具有生物学意义的特定蛋白质纳入目标离子列表，用于后续的离线气相碎裂，该碎裂使用原始收集的LC馏分的一份等分试样，其中一份等分试样也用于自下而上分析。
