Keeping up with the bases: 'nextgen' approaches to proteomics

著名的生物化学家亚瑟·科恩伯格在他的书《为了对酶的热爱》中曾经说过：“DNA和RNA是剧本，但蛋白质是演员”。蛋白质组学是对行为者的研究（无论是在独白中还是在人群场景中），在诊断学、新疾病生物标志物的分析、理解实现基因“意图”的基本方式方面具有巨大的潜力。目前，全球对蛋白质的研究（蛋白质组学）落后于我们对基因组和RNA的理解，如果没有激进的新技术方法，充分利用分析能力并将其与新的样品递送方法相结合，差距可能会扩大。挑战是多方面的。首先，一个真正的全局蛋白质组分析必须能够处理高度复杂的蛋白质混合物，一些蛋白质大量存在，另一些蛋白质的含量低得几乎可以忽略不计。这将需要一定程度的标准化，其中将低丰度蛋白质以足够的量带入分析步骤以进行分析，并且非选择性地对高丰度蛋白质进行采样。其次，目前的蛋白质组学仍然主要基于使用来自肠道的酶（胰蛋白酶）将蛋白质预先消化成多个较小的肽。这不仅使分析物的复杂性增加了约50倍，而且还掩盖了蛋白质世界的许多微妙之处（就像一堆砖块无法告知许多建筑类型和变体的结构一样，这些砖块可能是由这些砖块制成的）。未来的解决方案应该基于蛋白质水平的分析--建筑与其说是对砖块的研究，不如说是对砖块组装而成的整个结构的探索和庆祝。最后，我们通过相当麻烦的色谱法缓慢地（每个样品1- 4小时）递送肽。需要蛋白质水平递送的替代方法。我建议，我们应该计划分析蛋白质组，而不需要消化或色谱的复杂性。这带来了新的挑战，因为我们不能期望质谱仪能够一次分析整个蛋白质组（它太复杂了）。因此，我希望设计一种全新的蛋白质组分析方法，该方法基于在任何时间将少量蛋白质递送到分析平台。工业合作者发明了新型的基因改变蛋白质（“Affimers”，因为它们对选定的靶蛋白具有高亲和力），能够选择性地结合并一次钓出几个蛋白质。通过适当的分析仪器，我们应该能够以这样一种方式提供有效载荷（蛋白质），我们可以直接分析它们，捕获蛋白质世界的所有复杂性-建筑师的观点。从长远来看，有效载荷递送的工程解决方案可能使其成为蛋白质组分析的首选方法。

A famous biochemist, Arthur Kornberg, in his book "For the Love of Enzymes" once said 'DNA and RNA are the script, but proteins are the actors'. Proteomics, the study of the actors, (whether in soliloquy or in crowd scenes) has terrific potential in diagnostics, in the analysis of new disease biomarkers, in understanding the fundamental ways by which the 'intent' of the genes is realised. At present, the global study of proteins (proteomics) is lagging behind our understanding of genomes and RNA, and without radical new technical approaches, taking the best of the analytical capability and coupling it to new methods of sample delivery, the gap is likely to widen. The challenges are several. First, a true global proteome analysis has to be able to deal with a highly complex mixture of proteins, some present in huge quantities, others at vanishingly low levels. This will require a degree of normalization, in which low abundance proteins are brought to the analytical step in sufficient amounts for analysis, and high abundant proteins are non-selectively sampled. Secondly, current proteomics is still predominantly based on prior digestion of proteins to multiple smaller peptides using an enzyme (trypsin) derived from the gut. This not only increases analyte complexity about 50 times but also conceals much of the subtlety of the protein world (just as a pile of bricks cannot inform about the structure of the the many building types and variants that could have been made from those bricks). A future solution should be based on protein-level analysis - architecture is less about the study of bricks than it is the exploration and celebration of the entire structures that the bricks are assembled to create. Finally, we deliver peptides slowly (1-4h per sample) by rather troublesome chromatography. Alternative approaches to protein-level delivery are required. I propose that we should plan to analyse a proteome without the complication of digestion or of chromatography. This poses new challenges, because we cannot expect the mass spectrometer to be able to analyse a whole proteome at once (it is just too complex). I therefore wish to devise an entirely new approach to proteome analysis based on delivery of a small number of proteins at any one time to the analytical platforms. The industrial collaborator has invented new types of genetically altered proteins ('Affimers', because they have a high affinity for selected target proteins) that are capable of selectively binding and fishing out a few proteins at a time. With appropriate analytical instrumentation, we should then be able to deliver the payload (proteins) in such a way that we can analyse them directly, capturing all of the complexity of the protein world - the architect's view. In the longer term, engineering solutions to payload delivery could make this the preferred approach to proteome analysis.