High-Sensitivity LC-MS/MS Quantification of Peptides and Proteins in Complex Biological Samples: The Impact of Enzymatic Digestion and Internal Standard Selection on Method Performance
High-Sensitivity LC-MS/MS Quantification of Peptides and Proteins in Complex Biological Samples: The Impact of Enzymatic Digestion and Internal Standard Selection on Method Performance
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DOI：
10.1021/ac4015116
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发表时间：
2013-10-15
影响因子：
7.4
通讯作者：
van de Merbel, Nico C.
中科院分区：
文献类型：
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作者：
Bronsema, Kees J.;Bischoff, Rainer;van de Merbel, Nico C.
肽和蛋白质定量的两个重要方面，通过LC-MS/MS，酶消化步骤和内部标准化方法，进行了系统的研究与一个小的蛋白质，鲑鱼降钙素，可以分析没有和消化。从血浆中固相提取后，未消化的鲑鱼降钙素的定量导致10 pg/mL的定量下限，而引入胰蛋白酶消化步骤，随后定量特征肽，将其增加至50 pg/mL。与未消化的鲑鱼降钙素相比，消化导致样品复杂性增加，并且特征肽的选择性反应监测（SRM）转换选择性较低，因此特征肽的选择性反应监测（SRM）转换的干扰降低了灵敏度。比较了8种内标方法在有和没有消化的工作流程中的准确度和精密度。评价了标记物和稳定同位素标记（SIL）内标物，包括内部生成的O-18标记肽、可裂解SIL肽和通过特征肽的差示衍生化生成的内标物。我们得出结论，对于有和没有消化的工作流程，最好的内标物是分析物的SIL形式，尽管使用几种SIL特征肽和差异衍生的特征肽也导致方法的性能符合FDA指南。
Two important aspects of peptide and protein quantification by LC-MS/MS, the enzymatic digestion step and the internal standardization approach, were systematically investigated with a small protein, salmon calcitonin, which could be analyzed both without and with digestion. Quantification of undigested salmon calcitonin, after solid-phase extraction from plasma, resulted in a lower limit of quantification of 10 pg/mL, while introduction of a tryptic digestion step, followed by quantification of a signature peptide, increased this to 50 pg/mL. The sensitivity was reduced by interferences in the selected reaction monitoring (SRM) transition of the signature peptide due to the increase in sample complexity caused by the digestion and a less selective SRM transition of the signature peptide as compared to undigested salmon calcitonin. Eight internal standardization approaches were compared with respect to accuracy and precision in workflows with and without digestion. Analogue and stable-isotope-labeled (SIL) internal standards were evaluated including an in-house created O-18-labeled peptide, a cleavable SIL peptide, and an internal standard created by differential derivatization of the signature peptide. We conclude that the best internal standard for the workflows both with and without digestion was the SIL form of the analyte, although the use of several SIL signature peptides and a differentially derivatized signature peptide also resulted in methods with performances which meet the FDA guidelines.