Kerr Caitlin M, Schneider Olivia L, Tichy Shane, Huge Bonnie Jaskowski, Champion Matthew M
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
Berthiaume Institute for Precision Health, University of Notre Dame, Notre Dame, Indiana 46556, United States.
Anal Chem. 2025 Jan 14;97(1):649-657. doi: 10.1021/acs.analchem.4c05010. Epub 2025 Jan 1.
Intact protein analysis using mass spectrometry (MS) is an important technique to characterize and provide a comprehensive overview of protein complexity. It is also the basis of "top-down" approaches in proteomics to describe the proteoforms of single protein's post-translational modifications (PTMs). MS-based analysis of intact proteins benefits from high-resolution separations prior to electrospray ionization. Capillary isoelectric focusing (cIEF) is a high-resolution separation for proteins and peptides which is capable of separating proteoforms. MS detection coupled to cIEF can separate, detect, and characterize proteoforms at the molecular level. However, cIEF with MS detection is a compromised process. The concentration of ampholytes required for cIEF is mutually exclusive with mass spectrometer contamination. We have improved an online cIEF-ESI-MS interface to reduce (desalt) amino acid ampholytes in-line after cIEF and prior to electrospray ionization. In proof of principle experiments, >90% increase in area under the curve of the electropherograms was observed with the interface compared to without the interface. Protein standards including proteoforms of cytochrome C, myoglobin, and α-casein were separated and resolved with high reproducibility. The interface did not compromise the linearity of the cIEF pH gradient separations, achieving a high linearity with a of 0.99. In addition, a tryptic digest of BSA demonstrates baseline resolution of peptides with as little as 0.02 pI unit difference and a full width at half-maximum average of 7.1 s.
使用质谱（MS）进行完整蛋白质分析是一种重要技术，可用于表征蛋白质复杂性并提供全面概述。它也是蛋白质组学中“自上而下”方法的基础，用于描述单个蛋白质翻译后修饰（PTM）的蛋白质变体。基于MS的完整蛋白质分析受益于电喷雾电离之前的高分辨率分离。毛细管等电聚焦（cIEF）是一种用于蛋白质和肽的高分辨率分离技术，能够分离蛋白质变体。与cIEF联用的MS检测可以在分子水平上分离、检测和表征蛋白质变体。然而，cIEF与MS检测联用是一个存在问题的过程。cIEF所需两性电解质的浓度与质谱仪污染相互排斥。我们改进了一种在线cIEF-ESI-MS接口，以在cIEF之后和电喷雾电离之前在线减少（脱盐）氨基酸两性电解质。在原理验证实验中，与没有该接口相比，使用该接口观察到电泳图曲线下面积增加了90%以上。包括细胞色素C、肌红蛋白和α-酪蛋白蛋白质变体在内的蛋白质标准品被分离并以高重现性解析。该接口没有损害cIEF pH梯度分离的线性，实现了高达0.99的高线性。此外，牛血清白蛋白的胰蛋白酶消化显示出肽的基线分辨率，肽之间的等电点差异低至0.02个单位，半高宽平均为7.1秒。
