OncoProteomics Laboratory, Department of Medical Oncology, VU Medical Center, Amsterdam, The Netherlands; Department of Molecular and Cellular Neurobiology, Center for Medical Systems Biology/Neurogenomics and Cognitive Research, Vrije Universteit, Amsterdam, The Netherlands.
Proteomics Clin Appl. 2007 Jun;1(6):598-604. doi: 10.1002/prca.200600483. Epub 2007 May 11.
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 ˜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® 96, the whole serum peptide capture procedure is high-throughput (˜20 min per isolation of 96 samples in parallel), thereby facilitating large-scale disease profiling studies.
基于 MS 的血清肽谱分析是一种用于疾病诊断和生物标志物发现的新兴方法。最近已经引入了一种基于磁珠的离线血清肽捕获方法，与 MALDI-TOF-MS 相结合。然而，这些试剂尚未向广大科学界提供。在这里，我们开发了一种使用新型磁性 C18 珠进行血清肽捕获的方案，并在高通量磁性颗粒处理器上自动进行该程序。我们研究了珠平衡、肽结合和肽洗脱条件。该方法根据对照血清中的峰计数和离子强度的重现性进行评估。总体而言，这里报道的基于 DynaBead-RPC18 的血清样品处理方案具有重现性、稳健性，并且允许检测到 1 小时凝固的血清中 m/z 800-4000 范围内的约 200 个肽。粗血清和 0.5%TFA/0.15%正辛基葡萄糖处理的血清中归一化离子强度的平均内实验 %CV 分别为 12%（范围为 2-38%）和 10%（3-21%），而外实验 %CV 分别为 24%（10-53%）和 31%（10-59%）。重要的是，任何拥有 MALDI-TOF 仪器的人都可以使用这种方法进行血清肽谱分析。与 KingFisher®96 一起，整个血清肽捕获过程具有高通量（平行分离 96 个样本时约 20 分钟），从而促进了大规模疾病分析研究。
