Flensburg John, Haid Daniel, Blomberg Johan, Bielawski Jacek, Ivansson Daniel
Amersham Biosciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden.
J Biochem Biophys Methods. 2004 Sep 30;60(3):319-34. doi: 10.1016/j.jbbm.2004.01.010.
A new matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI-ToF MS), developed specifically for the identification and characterization of proteins and peptides in proteomic investigations, is described. The mass spectrometer which can be integrated with the 2-D gel electrophoresis workflow is a bench-top instrument, enabling rapid, reliable and unattended protein identification in low-, as well as high-throughput proteomics applications. To obtain precise information on peptide sequences, the instrument utilizes a timed ion gate and a unique quadratic field reflectron (Z2 technology), allowing single-run, post-source decay (PSD) of selected peptides. In this study, the performance of the instrument in reflectron, PSD and linear mode, respectively, was investigated. The results showed that the limit of detection for a single peptide in reflectron mode was 125 amol with a signal to noise ratio exceeding 20. Average mass resolution for peptides larger than 2000 u was around 13,000 full width, half maximum (FWHM). The limit for protein identification during peptide mass fingerprinting (PMF) was 500 amol with a sequence coverage of 18%. Mass error during PMF analysis was less than 15 ppm for 17 out of 25 (68%) identified peptides. In PSD mode, a complete series of y-ions of a CAF-derivatized peptide could be obtained from 3.75 fmol of material. The average mass error of PSD-generated fragments was less than 0.14 u. Finally, in linear mode, intact proteins with molecular masses greater than 300,000 u were detected with mass errors below 0.2%.
本文介绍了一种专门为蛋白质组学研究中蛋白质和肽段的鉴定与表征而开发的新型基质辅助激光解吸/电离飞行时间质谱仪（MALDI-ToF MS）。该质谱仪可与二维凝胶电泳工作流程集成，是一种台式仪器，能够在低通量和高通量蛋白质组学应用中实现快速、可靠且无人值守的蛋白质鉴定。为了获得肽段序列的精确信息，该仪器采用了定时离子门和独特的二次场反射器（Z2技术），允许对选定肽段进行单次运行的源后衰变（PSD）。在本研究中，分别对该仪器在反射模式、PSD模式和线性模式下的性能进行了研究。结果表明，在反射模式下，单个肽段的检测限为125 amol，信噪比超过20。大于2000 u的肽段的平均质量分辨率约为13000半高宽（FWHM）。肽质量指纹图谱（PMF）分析中蛋白质鉴定的下限为500 amol，序列覆盖率为18%。在25个已鉴定肽段中的17个（68%）中，PMF分析期间的质量误差小于15 ppm。在PSD模式下，从3.75 fmol的材料中可以获得CAF衍生肽段的完整y离子系列。PSD产生的碎片的平均质量误差小于0.14 u。最后，在线性模式下，检测到分子量大于300,000 u的完整蛋白质，质量误差低于0.2%。
