AIMMS Division of BioMolecular Analysis, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
J Proteome Res. 2013 Mar 1;12(3):1067-77. doi: 10.1021/pr301201x. Epub 2013 Feb 15.
Several proteomics approaches are available that are defined by the level (protein or peptide) at which analysis takes place. The most widely applied method still is bottom-up proteomics where the protein is digested into peptides that can be efficiently analyzed with a wide range of LC-MS or MALDI-TOF-MS instruments. Sample preparation for bottom-up proteomics experiments requires several treatment steps in order to get from the protein to the peptide level and can be very laborious. The most crucial step in such approaches is the protein digestion, which is often the bottleneck in terms of time consumption. Therefore, a significant gain in throughput may be obtained by speeding up the digestion process. Current techniques allow for reduction of the digestion time from overnight (~15 h) to minutes or even seconds. This advancement also makes integration into online systems feasible, thereby reducing the number of tedious sample handling steps and the risk of sample loss. In this review, an overview is given of the currently available digestion strategies and recent developments in the acceleration of the digestion process. Additionally, tailored approaches for classes of proteins that pose specific challenges are discussed.
有几种蛋白质组学方法，它们是根据分析发生的水平（蛋白质或肽）来定义的。应用最广泛的方法仍然是自上而下的蛋白质组学，其中蛋白质被消化成肽，然后可以使用广泛的 LC-MS 或 MALDI-TOF-MS 仪器对其进行有效分析。自下而上的蛋白质组学实验的样品制备需要几个处理步骤，才能从蛋白质水平到肽水平，这可能非常繁琐。在这些方法中，最关键的步骤是蛋白质消化，这通常是耗时的瓶颈。因此，通过加快消化过程可以显著提高通量。目前的技术可以将消化时间从过夜（约 15 小时）缩短到几分钟甚至几秒钟。这一进展也使得在线系统的集成成为可能，从而减少了繁琐的样品处理步骤和样品损失的风险。在这篇综述中，概述了目前可用的消化策略以及加速消化过程的最新进展。此外，还讨论了针对特定挑战的蛋白质类别的定制方法。
