Garaguso Ignazio, Borlak Jürgen
Department of Drug Research and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany.
Proteomics. 2008 Jul;8(13):2583-95. doi: 10.1002/pmic.200701147.
The analytical performance of MALDI-MS is highly influenced by sample preparation and the choice of matrix. Here we present an improved MALDI-MS sample preparation method for peptide mass mapping and peptide analysis, based on the use of the 2,5-dihydroxybenzoic acid matrix and prestructured sample supports, termed: matrix layer (ML). This sample preparation is easy to use and results in a rapid automated MALDI-MS and MS/MS with high quality spectra acquisition. The between-spot variation was investigated using standard peptides and statistical treatment of data confirmed the improvement gained with the ML method. Furthermore, the sample preparation method proved to be highly sensitive, in the lower-attomole range for peptides, and we improved the performance of MALDI-MS/MS for characterization of phosphopeptides as well. The method is versatile for the routine analysis of in-gel tryptic digests thereby allowing for an improved protein sequence coverage. Furthermore, reliable protein identification can be achieved without the need of desalting sample preparation. We demonstrate the performance and the robustness of our method using commercially available reference proteins and automated MS and MS/MS analyses of in-gel digests from lung tissue lysate proteins separated by 2-DE.
基质辅助激光解吸电离质谱（MALDI-MS）的分析性能受样品制备和基质选择的影响很大。本文基于使用2,5-二羟基苯甲酸基质和预结构化样品支持物（称为基质层，ML），提出了一种改进的用于肽质量图谱分析和肽分析的MALDI-MS样品制备方法。这种样品制备方法易于使用，可实现快速自动化的MALDI-MS和MS/MS分析，并能高质量采集光谱。使用标准肽对斑点间差异进行了研究，数据的统计处理证实了ML方法所取得的改进。此外，该样品制备方法具有高度灵敏性，对肽的检测限低至阿托摩尔级别，我们还提高了MALDI-MS/MS对磷酸化肽进行表征的性能。该方法对于凝胶内胰蛋白酶消化产物的常规分析具有通用性，从而能够提高蛋白质序列覆盖率。此外，无需对样品制备进行脱盐处理即可实现可靠的蛋白质鉴定。我们使用市售参考蛋白质以及对通过二维电泳分离的肺组织裂解物蛋白质的凝胶内消化产物进行自动化MS和MS/MS分析，展示了我们方法的性能和稳健性。
