Automated-serum peptide profiling using novel magnetic C18 beads off-line coupled to MALDI-TOF-MS
Automated-serum peptide profiling using novel magnetic C18 beads off-line coupled to MALDI-TOF-MS
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DOI：
10.1002/prca.200600483
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发表时间：
2007-06-01
影响因子：
2
通讯作者：
Li, Ka Wan
中科院分区：
文献类型：
--
作者：
Jimenez, Connie R.;El Filali, Zineb;Li, Ka Wan
质谱分析是一种新兴的疾病诊断和生物标志物发现方法。最近介绍了一种基于磁珠的离线血清肽捕获与MALDI-TOF-MS耦合的方法。然而，这些试剂对一般科学界来说是不可用的。在这里，我们开发了一种使用新型磁性C18微珠捕获血清肽的方案，并在高通量磁颗粒处理器上实现了该过程的自动化。我们研究了头平衡、肽结合和肽洗脱条件。根据对照血清中离子强度的峰计数和再现性对该方法进行了评价。总体而言，本文报道的基于dynabead - rpc18的血清样品处理方案具有可重复性和可靠性，并允许在凝固1小时的血清m/z 800-4000下检测类似200条肽。粗血清和0.5% TFA/0.15% n-辛基葡萄糖苷处理血清的标准化离子强度的平均实验内%CV分别为12%（范围2-38%）和10%(3-21%)，实验间%CV分别为24%（10-53%）和31%（10-59%）。重要的是，该方法可用于血清肽分析的任何人拥有MALDI-TOF仪器。结合KingFisher (R) 96，全血清肽捕获程序是高通量的（类似于96个样本的每次分离20分钟），从而促进了大规模的疾病分析研究。
Serum peptide profiling by MS is an emerging approach for disease diagnosis and biomarker discovery. A magnetic bead-based method for off-line serum peptide capture coupled to MALDI-TOF-MS has been recently introduced. However, the reagents are not available to the general scientific community. Here, we developed a protocol for serum peptide capture using novel magnetic C18 beads, and automated the procedure on a high-throughput magnetic particle processor. We investigated bead equilibration, peptide binding and peptide elution conditions. The method is evaluated in terms of peaks counts and reproducibility of ion intensities in control serum. Overall, the DynaBead-RPC18-based serum sample processing protocol reported here is reproducible, robust and allows for the detection of similar to 200 peptides at m/z 800-4000 of serum that was allowed to clot for 1 h. The average intra-experiment %CV of normalized ion intensities for crude serum and 0.5% TFA/0.15% n-octyl glucoside-treated serum, respectively, were 12% (range 2-38%) and 10% (3-21%) and the inter-experiment %CVs were 24% (10-53%) and 31% (10-59%). Importantly, this method can be used for serum peptide profiling by anyone in possession of a MALDI-TOF instrument. In conjunction with the KingFisher (R) 96, the whole serum peptide capture procedure is high-throughput (similar to 20 min per isolation of 96 samples in parallel), thereby facilitating large-scale disease profiling studies.