Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32304, United States.
Bruker Daltonics, Billerica, Massachusetts 01821, United States.
J Am Soc Mass Spectrom. 2023 Oct 4;34(10):2232-2246. doi: 10.1021/jasms.3c00187. Epub 2023 Aug 28.
"Top-down" proteomics analyzes intact proteins and identifies proteoforms by their intact mass as well as the observed fragmentation pattern in tandem mass spectrometry (MS/MS) experiments. Recently, hybrid ion mobility spectrometry-mass spectrometry (IM/MS) methods have gained traction for top-down experiments, either by allowing top-down analysis of individual isomers or alternatively by improving signal/noise and dynamic range for fragment ion assignment. We recently described the construction of a tandem-trapped ion mobility spectrometer/mass spectrometer (tandem-TIMS/MS) coupled with an ultraviolet (UV) laser and demonstrated a proof-of-principle for top-down analysis by UV photodissociation (UVPD) at 2-3 mbar. The present work builds on this with an exploration of a top-down method that couples tandem-TIMS/MS with UVPD and parallel-accumulation serial fragmentation (PASEF) MS/MS analysis. We first survey types and structures of UVPD-specific fragment ions generated in the 2-3 mbar pressure regime of our instrument. Notably, we observe UVPD-induced fragment ions with multiple conformations that differ from those produced in the absence of UV irradiation. Subsequently, we discuss how MS/MS spectra of top-down fragment ions lend themselves ideally for probability-based scoring methods developed in the bottom-up proteomics field and how the ability to record automated PASEF-MS/MS spectra resolves ambiguities in the assignment of top-down fragment ions. Finally, we describe the coupling of tandem-TIMS/MS workflows with UVPD and PASEF-MS/MS analysis for native top-down protein analysis.
“自上而下”的蛋白质组学分析完整的蛋白质，并根据其完整质量以及串联质谱（MS/MS）实验中观察到的碎裂模式来鉴定蛋白质形式。最近，混合离子淌度谱-质谱（IM/MS）方法在自上而下的实验中得到了应用，要么通过允许对单个异构体进行自上而下的分析，要么通过提高碎片离子分配的信号/噪声比和动态范围。我们最近描述了串联捕获离子淌度谱-质谱仪（串联-TIMS/MS）的构建，该仪器与紫外（UV）激光耦合，并通过在 2-3 mbar 下进行紫外光解（UVPD）证明了自上而下分析的原理。本工作在此基础上进行了探索，即将串联-TIMS/MS 与 UVPD 和并行积累串联碎裂（PASEF）MS/MS 分析相结合的自上而下方法。我们首先调查了在我们仪器的 2-3 mbar 压力范围内生成的 UVPD 特异性碎片离子的类型和结构。值得注意的是，我们观察到具有多个构象的 UVPD 诱导的碎片离子，这些构象与在没有紫外辐照的情况下产生的碎片离子不同。随后，我们讨论了自上而下的碎片离子的 MS/MS 谱如何非常适合在 Bottom-up 蛋白质组学领域开发的基于概率的评分方法，以及记录自动 PASEF-MS/MS 谱的能力如何解决自上而下的碎片离子分配的歧义。最后，我们描述了将串联-TIMS/MS 工作流程与 UVPD 和 PASEF-MS/MS 分析相结合，用于天然的自上而下的蛋白质分析。
