Protein Function Group, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK.
Methods. 2011 Aug;54(4):351-60. doi: 10.1016/j.ymeth.2011.05.005. Epub 2011 Jun 6.
Virtually all mass spectrometric-based methods for quantitative proteomics are at the peptide level, whether label-mediated or label-free. Absolute quantification in particular is based on the measurement of limit peptides, defined as those peptides that cannot be further fragmented by the protease in use. Complete release of analyte and (stable isotope labelled) standard ensures that the most reliable quantification data are recovered, especially when the standard peptides are in a different primary sequence context, such as sometimes occurs in the QconCAT methodology. Moreover, in label-free methods, incomplete digestion would diminish the ion current attributable to limit peptides and lead to artifactually low quantification data. It follows that an essential requirement for peptide-based absolute quantification in proteomics is complete and consistent proteolysis to limit peptides. In this paper we describe strategies to assess completeness of proteolysis and discuss the potential for variance in digestion efficiency to compromise the ensuing quantification data. We examine the potential for kinetically favoured routes of proteolysis, particularly at the last stages of the digestion, to direct products into 'dead-end' mis-cleaved products.
实际上，所有基于质谱的定量蛋白质组学方法都是在肽水平上进行的，无论是基于标记的方法还是无标记的方法。特别是绝对定量是基于对限定肽的测量，限定肽被定义为那些不能被使用的蛋白酶进一步片段化的肽。分析物和（稳定同位素标记的）标准品的完全释放确保了最可靠的定量数据的恢复，特别是当标准肽在不同的一级序列背景下时，例如在 QconCAT 方法中有时会发生。此外，在无标记方法中，不完全的消化会降低归因于限定肽的离子电流，并导致定量数据人为地降低。因此，蛋白质组学中基于肽的绝对定量的一个基本要求是对限定肽进行完全和一致的蛋白水解。在本文中，我们描述了评估蛋白水解完全性的策略，并讨论了消化效率的差异可能会影响随后的定量数据的情况。我们研究了动力学有利的蛋白水解途径的可能性，特别是在消化的最后阶段，将产物导向“死胡同”的错误切割产物。
