Department of Chemistry , University of Wisconsin-Madison , Madison , Wisconsin 53706 , United States.
Human Proteomics Program , University of Wisconsin-Madison , Madison , Wisconsin 53705 , United States.
Anal Chem. 2019 Mar 19;91(6):3835-3844. doi: 10.1021/acs.analchem.8b04082. Epub 2019 Feb 25.
Mass spectrometry (MS) based top-down proteomics provides rich information about proteoforms arising from combinatorial amino acid sequence variations and post-translational modifications (PTMs). Fourier transform ion cyclotron resonance (FT-ICR) MS affords ultrahigh resolving power and provides high-accuracy mass measurements, presenting a powerful tool for top-down MS characterization of proteoforms. However, the detection and characterization of large proteins from complex mixtures remain challenging due to the exponential decrease in S: N with increasing molecular weight (MW) and coeluting low-MW proteins; thus, size-based fractionation of complex protein mixtures prior to MS analysis is necessary. Here, we directly combine MS-compatible serial size exclusion chromatography (sSEC) fractionation with 12 T FT-ICR MS for targeted top-down characterization of proteins from complex mixtures extracted from human and swine heart tissue. Benefiting from the ultrahigh resolving power of FT-ICR, we isotopically resolved 31 distinct proteoforms (30-50 kDa) simultaneously in a single mass spectrum within a 100 m/ z window. Notably, within a 5 m/ z window, we obtained baseline isotopic resolution for 6 distinct large proteoforms (30-50 kDa). The ultrahigh resolving power of FT-ICR MS combined with sSEC fractionation enabled targeted top-down analysis of large proteoforms (>30 kDa) from the human heart proteome without extensive chromatographic separation or protein purification. Further separation of proteoforms inside the mass spectrometer (in-MS) allowed for isolation of individual proteoforms and targeted electron capture dissociation (ECD), yielding high sequence coverage. sSEC/FT-ICR ECD facilitated the identification and sequence characterization of important metabolic enzymes. This platform, which facilitates deep interrogation of proteoform primary structure, is highly tunable, allows for adjustment of MS and MS/MS parameters in real time, and can be utilized for a variety of complex protein mixtures.
基于质谱（MS）的自上而下蛋白质组学提供了丰富的信息，这些信息来自组合氨基酸序列变化和翻译后修饰（PTM）产生的蛋白质形式。傅里叶变换离子回旋共振（FT-ICR）MS 提供了超高分辨率，并提供了高精度的质量测量，为蛋白质形式的自上而下 MS 表征提供了强大的工具。然而，由于分子量（MW）增加时 S: N 呈指数下降，以及共洗脱的低 MW 蛋白质，从复杂混合物中检测和表征大蛋白质仍然具有挑战性；因此，在进行 MS 分析之前，需要对复杂蛋白质混合物进行基于大小的分级。在这里，我们直接将 MS 兼容的串联排阻色谱（sSEC）分级与 12 T FT-ICR MS 相结合，用于对从人心脏和猪心脏组织中提取的复杂混合物中的蛋白质进行靶向自上而下的表征。得益于 FT-ICR 的超高分辨率，我们在 100 m/ z 窗口内的单个质谱中同时同位素分辨了 31 个不同的蛋白质形式（30-50 kDa）。值得注意的是，在 5 m/ z 窗口内，我们获得了 6 个不同大蛋白质形式（30-50 kDa）的基线同位素分辨率。FT-ICR MS 与 sSEC 分级相结合的超高分辨率，使我们能够在无需广泛的色谱分离或蛋白质纯化的情况下，对人心脏蛋白质组中的大蛋白质形式（>30 kDa）进行靶向自上而下的分析。在质谱仪内部对蛋白质形式进行进一步分离（in-MS）允许分离单个蛋白质形式并进行靶向电子捕获解离（ECD），从而获得高序列覆盖率。sSEC/FT-ICR ECD 促进了重要代谢酶的鉴定和序列特征分析。该平台促进了对蛋白质一级结构的深入研究，具有高度可调性，允许实时调整 MS 和 MS/MS 参数，并可用于各种复杂的蛋白质混合物。
