Zheng Jing, Li Nan, Ridyard Marc, Dai Hui, Robbins Stephen M, Li Liang
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
J Proteome Res. 2005 Sep-Oct;4(5):1709-16. doi: 10.1021/pr050157w.
Recent advances in MALDI MS/MS instrumentation allow a high degree of automation in the efficient detection of peptide fragment ions that can be used for protein identification. However, the performance of the technique is dependent on the MALDI sample preparation. We present a simple and robust two-layer sample preparation method tailored for sensitive and reproducible generation of MALDI MS/MS data. This method produces a strong and uniform crystal layer which allows acquisition of high quality MS/MS spectra over the entire sample surface area. Furthermore, due to its crystal strength, the matrix/sample layer can be washed extensively on target, enabling direct analysis of samples containing impurities, such as salts and surfactants. This method is demonstrated to be very useful in routine analysis of in-gel tryptic digests of silver-stained protein gel spots, without the need of desalting steps or hunting for "hot" spots. As an example, seven threonine-phosphorylated proteins involved in signal transduction in response to growth factor stimulation within the lipid raft fractions of the IMR5 neuroblastoma cells have been identified using differential gel display, in-gel digestion and MALDI MS/MS with the new two-layer sample preparation method. Some of these proteins have the functions of maintaining raft structure or cell signaling.
基质辅助激光解吸电离串联质谱（MALDI MS/MS）仪器的最新进展使得在高效检测可用于蛋白质鉴定的肽片段离子方面实现了高度自动化。然而，该技术的性能取决于MALDI样品制备。我们提出了一种简单且稳健的两层样品制备方法，专为灵敏且可重复地生成MALDI MS/MS数据而定制。这种方法产生了一个强大且均匀的晶体层，可在整个样品表面积上采集高质量的MS/MS光谱。此外，由于其晶体强度，基质/样品层可在靶板上进行大量洗涤，从而能够直接分析含有杂质（如盐和表面活性剂）的样品。该方法在银染蛋白凝胶斑点的胶内胰蛋白酶消化产物的常规分析中非常有用，无需脱盐步骤或寻找“热点”。例如，使用差异凝胶电泳、胶内消化以及采用新的两层样品制备方法的MALDI MS/MS，已鉴定出IMR5神经母细胞瘤细胞脂筏部分中参与生长因子刺激响应信号转导的七种苏氨酸磷酸化蛋白。其中一些蛋白质具有维持脂筏结构或细胞信号传导的功能。
