Experimental Biological Sciences, Pfizer, Kent CT13 9NJ, UK.
Anal Biochem. 2010 Apr 15;399(2):202-10. doi: 10.1016/j.ab.2010.01.002. Epub 2010 Jan 11.
An immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantitation of the zinc endopeptidase matrix metalloproteinase 9 (MMP-9) from mouse serum. Sample preparation for the assay included magnetic bead-based enrichment using an MMP-9 antibody and was performed in a 96-well plate format using a liquid-handling robotic platform. The surrogate peptide GSPLQGPFLTAR derived from MMP-9 by trypsin digestion was monitored using an on-line capillary flow trap-release chromatography setup incorporating a series of trap columns (C18, strong cation exchange, and another C18) prior to nanoflow chromatography and nanospray ionization with selected reaction monitoring (SRM) detection. The assay was fit-for-purpose validated and found to be accurate (<15% interbatch relative error) and precise (<15% interbatch coefficient of variation) across a range from 0.03 to 7.3nM mouse MMP-9. Finally, the method was employed to measure MMP-9 concentrations in 30 naïve mouse serum samples, and results were compared with those obtained by an immunoassay.
建立了一种免疫亲和液相色谱-串联质谱（LC-MS/MS）方法，用于定量检测小鼠血清中的锌内肽酶基质金属蛋白酶 9（MMP-9）。该测定的样品制备包括使用 MMP-9 抗体进行基于磁珠的富集，并使用液体处理机器人平台在 96 孔板格式中进行。通过胰蛋白酶消化从 MMP-9 衍生的替代肽 GSPLQGPFLTAR，使用在线毛细管流阱释放色谱装置进行监测，该装置包含一系列阱柱（C18、强阳离子交换和另一个 C18），然后进行纳流色谱和纳喷雾电离，采用选择反应监测（SRM）检测。该测定方法经过适用性验证，发现其在 0.03 至 7.3nM 小鼠 MMP-9 的范围内具有准确性（批间相对误差<15%）和精密度（批间变异系数<15%）。最后，该方法用于测量 30 个未经处理的小鼠血清样本中的 MMP-9 浓度，并将结果与免疫测定法获得的结果进行比较。
