Chemistry Department, Indiana University, 800 E. Kirkwood Avenue, Bloomington, Indiana 47405, United States.
J Am Soc Mass Spectrom. 2024 Aug 7;35(8):1969-1975. doi: 10.1021/jasms.4c00222. Epub 2024 Jul 16.
High purity plasmid DNA is a raw material for recombinant protein production as well as an active ingredient in DNA vaccines. There are four primary plasmid structures that can be observed in a typical plasmid formulation: supercoiled, relaxed (circular), linearized, and condensed. Determining what structures are present in a sample is important, as the structure can affect activity; the supercoiled structure has the highest activity, and >90% supercoiled is desired for industry standards. Recently, charge detection mass spectrometry (CD-MS) was used to distinguish two of the structures, supercoiled and condensed, by measuring the charge deposited on the ions by positive mode electrospray. Here, CD-MS is used to probe the structures of DNA plasmids during compaction with polycations, and through enzymatic treatment to relax and linearize plasmids. We find that all four structural types for plasmid DNA have unique charging profiles that can be distinguished using CD-MS. The extent of mechanical shearing of the DNA plasmids during electrospray is strongly influenced by the structural type.
高纯度质粒 DNA 是重组蛋白生产的原料，也是 DNA 疫苗的有效成分。在典型的质粒制剂中，可以观察到四种主要的质粒结构：超螺旋、松弛（环形）、线性化和浓缩。确定样品中存在哪些结构很重要，因为结构会影响活性；超螺旋结构的活性最高，行业标准要求超过 90%的超螺旋。最近，通过测量正模式电喷雾在离子上沉积的电荷，电荷检测质谱（CD-MS）被用于区分两种结构，超螺旋和浓缩，。在这里，CD-MS 用于探测在与聚阳离子缩合过程中 DNA 质粒的结构，并通过酶处理来松弛和线性化质粒。我们发现，质粒 DNA 的所有四种结构类型都具有独特的电荷分布特征，可以通过 CD-MS 来区分。在电喷雾过程中，DNA 质粒的机械剪切程度受结构类型的强烈影响。
