Deshpande A S, Lin A, O'Bryon I, Aufrecht J A, Merkley E D
Biogeochemical Transformations Group, Pacific Northwest National Laboratory, Richland, Washington, USA.
Chemical and Biological Signatures Group, Pacific Northwest National Laboratory, Richland, Washington, USA.
Expert Rev Proteomics. 2025 Mar;22(3):89-106. doi: 10.1080/14789450.2025.2476979. Epub 2025 Apr 6.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been a leading method for proteomics for 30 years. Advantages provided by LC-MS/MS are offset by significant disadvantages, including cost. Recently, several non-mass spectrometric methods have emerged, but little information is available about their capacity to analyze the complex mixtures routine for mass spectrometry.
We review recent non-mass-spectrometric methods for sequencing proteins and peptides, including those using nanopores, sequencing by degradation, reverse translation, and short-epitope mapping, with comments on bioinformatics challenges, fundamental limitations, and areas where new technologies will be more or less competitive with LC-MS/MS. In addition to conventional literature searches, instrument vendor websites, patents, webinars, and preprints were also consulted to give a more up-to-date picture.
Many new technologies are promising. However, demonstrations that they outperform mass spectrometry in terms of peptides and proteins identified have not yet been published, and astute observers note important disadvantages, especially relating to the dynamic range of single-molecule measurements of complex mixtures. Still, even if the performance of emerging methods proves inferior to LC-MS/MS, their low cost could create a different kind of revolution: a dramatic increase in the number of biology laboratories engaging in new forms of proteomics research.
液相色谱-串联质谱法（LC-MS/MS）在过去30年一直是蛋白质组学的主要方法。LC-MS/MS的优势被包括成本在内的显著劣势所抵消。最近，出现了几种非质谱方法，但关于它们分析质谱常规复杂混合物能力的信息却很少。
我们综述了近期用于蛋白质和肽测序的非质谱方法，包括使用纳米孔的方法、降解测序、反向翻译和短表位图谱分析，并对生物信息学挑战、基本局限性以及新技术与LC-MS/MS相比或多或少具有竞争力的领域进行了评论。除了常规文献检索外，还查阅了仪器供应商网站、专利、网络研讨会和预印本，以提供更最新的情况。
许多新技术很有前景。然而，尚未发表关于它们在鉴定肽和蛋白质方面优于质谱的证明，敏锐的观察家指出了重要的劣势，特别是与复杂混合物单分子测量的动态范围有关。尽管如此，即使新兴方法的性能被证明不如LC-MS/MS，其低成本也可能引发另一种变革：从事新型蛋白质组学研究的生物学实验室数量大幅增加。
