Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
Anal Chem. 2012 Jul 17;84(14):6116-21. doi: 10.1021/ac300926h. Epub 2012 Jun 27.
We demonstrate the use of capillary zone electrophoresis with an electrokinetic sheath-flow electrospray interface coupled to a triple-quadrupole mass spectrometer for the accurate and precise quantification of Leu-enkephalin in a complex mixture using multiple-reaction monitoring (MRM). Assay time is <6 min, with no re-equilibration required between runs. A standard curve of Leu-enkephalin was performed in the presence of a background tryptic digest of bovine albumin. We demonstrate reasonably reproducible peak heights (21% relative standard deviation), retention times (better than 1% relative standard deviation), and robust electrospray quality. Our limit of detection (3σ) was 60 pM, which corresponds to the injection of 335 zmol of peptide. This is a 10-20-fold improvement in mass sensitivity than we have obtained by nano HPLC/MRM and substantially better than reported for LC/MS/MS. Further quantification was performed in the presence of stable-isotope-labeled versions of the peptides; under these conditions, linearity was observed across nearly 4 orders of magnitude. The concentration detection limit was 240 pM for the stable-isotope-labeled quantification.
我们展示了使用毛细管区带电泳与电动鞘流电喷雾接口结合三重四极杆质谱仪，通过多反应监测（MRM）对复杂混合物中的亮氨酸脑啡肽进行准确和精确的定量。测定时间<6 分钟，无需在运行之间重新平衡。在存在牛血清白蛋白的胰蛋白酶消化物的背景下进行亮氨酸脑啡肽的标准曲线。我们证明了相当可重现的峰高（21%相对标准偏差）、保留时间（优于 1%相对标准偏差）和稳健的电喷雾质量。我们的检测限（3σ）为 60 pM，这相当于注射 335 zmol 的肽。这比我们通过纳升级高效液相色谱/MRM 获得的质量灵敏度提高了 10-20 倍，并且大大优于 LC/MS/MS 的报道。进一步的定量是在肽的稳定同位素标记版本存在下进行的；在这些条件下，线性观察到近 4 个数量级。稳定同位素标记定量的浓度检测限为 240 pM。
