Garnett Graham A E, Starke Melissa J, Shaurya Alok, Li Janessa, Hof Fraser
Department of Chemistry, University of Victoria , Victoria, British Columbia V8W3 V6, Canada.
Anal Chem. 2016 Apr 5;88(7):3697-703. doi: 10.1021/acs.analchem.5b04508. Epub 2016 Mar 25.
Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods.
使用质谱对翻译后甲基化物种进行全蛋白质组研究，因样本多样性高、肽段之间的离子化竞争以及质量冗余而变得复杂。到目前为止，基于抗体的富集技术推动了甲基化蛋白质组学的发展，但现有泛特异性抗体的可靠性、泛特异性、多克隆性质和稳定性存在问题，无法为研究人员提供一个标准、可靠的平台。我们发明了一种阴离子超分子主体，它可以与含甲基赖氨酸的肽段选择性地形成主客体复合物，并利用它创建了一个甲基赖氨酸亲和柱。该柱基于甲基化分离肽段，这是同类商业阳离子交换柱无法实现的壮举。在进行标准蛋白质组学分析之前，将蛋白酶解的核提取物在甲基亲和柱上进行分离。该实验表明，这种化学甲基亲和柱能够富集并改善对复杂蛋白质混合物中甲基赖氨酸残基的分析。我们在生物分子驱动的富集方法背景下讨论了这一进展的重要性。
