Kin-Wing Lui, , , Sai-Ming Ngai, , and , Ting-Fung Chan*,
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引用次数: 0
摘要
电喷雾电离(ESI)-质谱(MS)是分析翻译后修饰蛋白的关键平台。随着质谱仪器和数据分析方法的不断进步,对完整的变形形态进行自上而下的分析已经变得非常可行。为了准确定量具有不同翻译后修饰(PTMs)的蛋白质形态,必须考虑PTMs对ESI-MS检测效率的影响。20年前,Kelleher及其同事提出在ESI-MS中使用蛋白离子相对比(PIRRs)和片段离子相对比(FIRRs)进行蛋白形态定量。虽然FIRR定量已经得到了广泛的研究,但PIRR定量的可靠性──特别是对于具有不同PTM程度的蛋白质形态──仍未得到充分的评估。在本研究中,我们使用多种位点特异性乙酰化重组组蛋白H3蛋白形式进一步验证了自上而下ESI-MS中PIRR定量的保真度。这些携带不同程度乙酰化的蛋白形式是通过在琥珀色终止密码子上加入乙酰赖氨酸的正交翻译系统产生的。紫外分光光度法绝对定量后,样品按等长比例混合,采用直接输注- esi - ms或弱阳离子交换/亲水性相互作用- esi - ms进行分析。我们的结果表明,无论乙酰化程度或位点如何,PIRRs都符合理论比率。这些发现加强了自顶向下的蛋白质形态定量的有效性,特别是对于组蛋白。
Evaluation of Protein Ion Relative Ratio Quantification in Top-down Electrospray Ionization-Mass Spectrometry Using Site-Specific Acetylated Recombinant Histone H3 Proteoforms
Electrospray ionization (ESI)-mass spectrometry (MS) is a key platform for analyzing post-translationally modified proteins. With continuous advances in MS instruments and data analysis methods, top-down analysis of intact proteoforms has become highly feasible. To accurately quantify proteoforms with varying post-translational modifications (PTMs), the influence of PTMs on the ESI-MS detection efficiency must be considered. Two decades ago, Kelleher and co-workers proposed using protein ion relative ratios (PIRRs) and fragment ion relative ratios (FIRRs) in ESI-MS for proteoform quantification. While FIRR quantification has been extensively studied, the reliability of PIRR quantification─particularly for proteoforms with varying PTM degrees─remains under-evaluated. In this study, we further validated the fidelity of PIRR quantification in top-down ESI-MS using various site-specifically acetylated recombinant histone H3 proteoforms. These proteoforms, carrying varied degrees of acetylation, were produced using an orthogonal translation system that incorporates acetyllysine at the amber stop codons. After absolute quantification by UV spectrophotometry, samples were mixed in isometric ratios and analyzed by either direct infusion-ESI-MS or weak cation exchange/hydrophilic interaction-ESI-MS. Our results show that PIRRs match theoretical ratios regardless of the acetylation degree or site. These findings reinforce the validity of top-down proteoform quantification, especially for histone proteins.
期刊介绍:
The Journal of the American Society for Mass Spectrometry presents research papers covering all aspects of mass spectrometry, incorporating coverage of fields of scientific inquiry in which mass spectrometry can play a role.
Comprehensive in scope, the journal publishes papers on both fundamentals and applications of mass spectrometry. Fundamental subjects include instrumentation principles, design, and demonstration, structures and chemical properties of gas-phase ions, studies of thermodynamic properties, ion spectroscopy, chemical kinetics, mechanisms of ionization, theories of ion fragmentation, cluster ions, and potential energy surfaces. In addition to full papers, the journal offers Communications, Application Notes, and Accounts and Perspectives