Kaulich Philipp T, Tholey Andreas
Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel, Germany.
Proteomics. 2025 Apr 27:e202400338. doi: 10.1002/pmic.202400338.
Manifold biological processes at all levels of transcription and translation can lead to the formation of a high number of different protein species (i.e., proteoforms), which outnumber the sequences encoded in the genome by far. Due to the large number of protein molecules formed in this way, which span an enormous range of different physicochemical properties, proteoforms are the functional drivers of all biological processes, creating the need for powerful analytical approaches to decipher this language of life. While bottom-up proteomics has become the most widely used approach, providing features such as high sensitivity, depth of analysis, and throughput, it has its limitations when it comes to identifying, quantifying, and characterizing proteoforms. In particular, the major bottleneck is to assign peptide-level information to the original proteoforms. In contrast, top-down proteomics (TDP) targets the direct analysis of intact proteoforms. Despite being characterized by a number of technological challenges, the TDP community has established numerous protocols that allow easy implementation in any proteomics laboratory. In this viewpoint, we compare both approaches, argue that it is worth embedding TDP experiments, and show fields of research in which TDP can be successfully implemented to perform integrative multi-level proteoformics.
转录和翻译各个层面的多种生物学过程可导致大量不同蛋白质种类（即蛋白质异构体）的形成，其数量远超基因组中编码的序列。由于以这种方式形成的蛋白质分子数量众多，涵盖了极为广泛的不同物理化学性质，蛋白质异构体是所有生物学过程的功能驱动因素，这就需要强大的分析方法来解读这种生命语言。虽然自下而上的蛋白质组学已成为使用最广泛的方法，具有高灵敏度、分析深度和通量等特点，但在识别、定量和表征蛋白质异构体方面存在局限性。特别是，主要瓶颈在于将肽段水平的信息与原始蛋白质异构体进行关联。相比之下，自上而下的蛋白质组学（TDP）旨在直接分析完整的蛋白质异构体。尽管面临诸多技术挑战，但TDP领域已建立了许多方案，便于在任何蛋白质组学实验室中实施。在本文观点中，我们比较了这两种方法，认为值得开展TDP实验，并展示了TDP能够成功实施以进行整合多层次蛋白质异构体组学研究的领域。
