Su Pei, McGee John P, Hollas Michael A R, Fellers Ryan T, Durbin Kenneth R, Greer Joseph B, Early Bryan P, Yip Ping F, Zabrouskov Vlad, Srzentić Kristina, Senko Michael W, Compton Philip D, Kelleher Neil L, Kafader Jared O
Departments of Molecular Biosciences, Chemistry and Chemical and Biological Engineering and the Feinberg School of Medicine, Northwestern University, Evanston, IL, USA.
ImmPro, Inc., Evanston, IL, USA.
Nat Protoc. 2025 Jan 2. doi: 10.1038/s41596-024-01091-y.
Individual ion mass spectrometry (IMS) is the Orbitrap-based extension of the niche mass spectrometry technique known as charge detection mass spectrometry (CDMS). While traditional CDMS analysis is performed on in-house-built instruments such as the electrostatic linear ion trap, IMS extends CDMS analysis to Orbitrap analyzers, allowing charge detection analysis to be available to the scientific community at large. IMS simultaneously measures the mass-to-charge ratios (m/z) and charges (z) of hundreds to thousands of individual ions within one acquisition event, creating a spectral output directly into the mass domain without the need for further spectral deconvolution. A mass distribution or 'profile' can be created for any desired sample regardless of composition or heterogeneity. To assist in reducing IMS analysis to practice, we developed this workflow for data acquisition and subsequent data analysis, which includes (i) protein sample preparation, (ii) attenuation of ion signals to obtain individual ions, (iii) the creation of a charge-calibration curve from standard proteins with known charge states and finally (iv) producing a meaningful mass spectral output from a complex or unknown sample by using the STORIboard software. This protocol is suitable for users with prior experience in mass spectrometry and bioanalytical chemistry. First, the analysis of protein standards in native and denaturing mode is presented, setting the foundation for the analysis of complex mixtures that are intractable via traditional mass spectrometry techniques. Examples of complex mixtures included here demonstrate the relevant analysis of an intact human monoclonal antibody and its intricate glycosylation patterns.
单离子质谱（IMS）是基于轨道阱的一种特殊质谱技术——电荷检测质谱（CDMS）的扩展。传统的CDMS分析是在诸如静电线性离子阱等自行搭建的仪器上进行的，而IMS将CDMS分析扩展到了轨道阱分析仪，使电荷检测分析能够为广大科学界所用。IMS在一次采集事件中同时测量数百至数千个单个离子的质荷比（m/z）和电荷数（z），直接生成进入质量域的光谱输出，无需进一步的光谱去卷积。可以为任何所需样品创建质量分布或“谱图”，而无需考虑其组成或异质性。为了帮助将IMS分析付诸实践，我们开发了这个用于数据采集和后续数据分析的工作流程，其中包括：（i）蛋白质样品制备；（ii）衰减离子信号以获得单个离子；（iii）用已知电荷状态的标准蛋白质创建电荷校准曲线；最后（iv）使用STORIboard软件从复杂或未知样品中生成有意义的质谱输出。本方案适用于有质谱和生物分析化学经验的用户。首先，介绍了天然和变性模式下蛋白质标准品的分析，为通过传统质谱技术难以分析的复杂混合物的分析奠定基础。这里包含的复杂混合物示例展示了完整人类单克隆抗体及其复杂糖基化模式的相关分析。
