Interdisciplinary Research Centre HALOmem, Charles Tanford Protein Center, Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Halle, Germany.
Interdisciplinary Research Centre HALOmem, Charles Tanford Protein Center, Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Halle, Germany.
J Proteomics. 2020 Jun 30;222:103793. doi: 10.1016/j.jprot.2020.103793. Epub 2020 Apr 27.
The activity of most proteins and protein complexes relies on the formation of defined three-dimensional structures. The analysis of these arrangements is therefore key for understanding their function and regulation in the cell. Besides the traditional structural techniques, structural mass spectrometry delivers insights into the various aspects of protein structure, including stoichiometry, protein-ligand interactions and solvent accessibility. The latter is usually obtained from labelling experiments. In this study, we evaluate two chemical labelling strategies using N-hydroxysuccinimidyl acetate and diethylpyrocarbonate as labelling reagents. We characterised the mass spectra of modified peptides and assessed labelling reactivity of individual amino acid residues in intact proteins. Importantly, we uncovered neutral losses from diethylpyrocarbonate modified amino acids improving the assignments of the peptide fragment spectra. We further established a quantitative labelling workflow to determine labelling percentage and unambiguously distinguish solvent accessible amino acid residues from stochastically labelled residues. Finally, we used ion mobility MS to explore whether labelled proteins maintain their structures and remain stable. We conclude that labelling using N-hydroxysuccinimidyl acetate and diethylpyrocarbonate delivers comparable results, however, N-hydroxysuccinimidyl acetate labelling is compatible with standard proteomic workflows while diethylpyrocarbonate labelling requires specialised experimental conditions and data analysis. SIGNIFICANCE: Covalent labelling is widely used to identify solvent accessible amino acid residues of proteins or protein complexes. However, with increasing sensitivity of available MS instrumentation, a high number of modified residues is usually observed making an unambiguous assignment of solvent accessible residues necessary. In this study, we establish a quantitative labelling workflow for two different labelling strategies to identify accessible amino acid residues. In addition, we characterise observed mass spectra of modified peptides and identified neutral loss of DEPC modified amino acid residues during HCD fragmentation improving their assignments.
大多数蛋白质和蛋白质复合物的活性依赖于形成明确的三维结构。因此，分析这些排列结构对于理解它们在细胞中的功能和调节至关重要。除了传统的结构技术外，结构质谱还提供了对蛋白质结构各个方面的深入了解，包括化学计量、蛋白质-配体相互作用和溶剂可及性。后者通常通过标记实验获得。在这项研究中，我们使用 N-羟基琥珀酰亚胺基乙酸酯和二乙基焦碳酸酯作为标记试剂，评估了两种化学标记策略。我们对修饰肽的质谱进行了特征描述，并评估了完整蛋白质中单个氨基酸残基的标记反应性。重要的是，我们从二乙基焦碳酸酯修饰的氨基酸中发现了中性损失，从而提高了肽片段谱的分配。我们进一步建立了一种定量标记工作流程，以确定标记百分比，并明确区分溶剂可及的氨基酸残基和随机标记的氨基酸残基。最后，我们使用离子淌度 MS 来探索标记后的蛋白质是否保持其结构并保持稳定。我们的结论是，使用 N-羟基琥珀酰亚胺基乙酸酯和二乙基焦碳酸酯进行标记可获得类似的结果，然而，N-羟基琥珀酰亚胺基乙酸酯标记与标准蛋白质组学工作流程兼容，而二乙基焦碳酸酯标记需要特殊的实验条件和数据分析。
共价标记广泛用于鉴定蛋白质或蛋白质复合物的溶剂可及氨基酸残基。然而，随着可用 MS 仪器的灵敏度不断提高，通常会观察到大量的修饰残基，因此有必要对溶剂可及的残基进行明确的分配。在这项研究中，我们建立了一种用于两种不同标记策略的定量标记工作流程，以鉴定可及的氨基酸残基。此外，我们还对修饰肽的观察到的质谱进行了特征描述，并在 HCD 碎裂过程中鉴定了二乙基焦碳酸酯修饰的氨基酸残基的中性损失，从而提高了它们的分配。
