Center for Bioinformatics-Junior Research Group for Protein-Protein Interactions and Computational Proteomics, Universität des Saarlandes, 66123 Saarbrücken, Germany.
Rapid Commun Mass Spectrom. 2010 Nov 30;24(22):3279-89. doi: 10.1002/rcm.4771.
Metal labelling of peptides and proteins using high-affinity metal-chelating compounds has found widespread applications in the medical and bioanalytical fields. In the present study we investigated the analysis of peptides derivatized either with cysteine- or amino group-directed metal-bound DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The metal complexes of DOTA were shown to be stable under MALDI-MS conditions. The introduction of the metal label led in a number of cases to significantly increased signal-to-noise (S/N) values and thus improved sensitivity of the labelled peptides compared to their unlabelled counterparts, especially for multiply labelled peptides. The presence of the labels did alter the tandem mass spectrometric (MS/MS) behaviour, namely the formation of sequence specific a-, b- and y-ion series, in dependence of the position of the label within the peptide sequence. For cysteine-derivatized peptides several label-specific reporter ions and characteristic immonium ions could be identified. Amino-directed labelling led only to the formation of characteristic immonium ions in ε-amino groups of lysine, whereas N-terminal labelling in some cases led to the formation of a(1)- and b(1)-ions. The results clearly show that MALDI-MS is suitable for the analysis of metal-labelled peptides, which was also confirmed in liquid chromatography (LC)/MALDI-based identification of proteins in a model protein mixture labelled with Cys-reactive DOTA. Here, in comparison to a run with alkylated cysteines, more than 50% more cysteine-containing peptides were identified.
使用高亲和性金属螯合化合物对肽和蛋白质进行金属标记，已在医学和生物分析领域得到广泛应用。本研究探讨了用半胱氨酸或氨基导向的金属结合 DOTA（1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸）螯合剂衍生的肽在基质辅助激光解吸/电离质谱（MALDI-MS）中的分析。研究表明，DOTA 的金属配合物在 MALDI-MS 条件下稳定。在许多情况下，引入金属标签会导致标记肽的信号与噪声（S/N）值显著增加，从而提高其灵敏度，与未标记肽相比，尤其是对多重标记肽。标签的存在确实改变了串联质谱（MS/MS）行为，即在标签在肽序列中的位置取决于标签，从而形成序列特异性 a-、b-和 y-离子系列。对于半胱氨酸衍生的肽，可以识别出几个标签特异性报告离子和特征的亚铵离子。氨基酸导向的标记仅导致赖氨酸ε-氨基中形成特征性的亚铵离子，而 N-末端标记在某些情况下会导致 a(1)-和 b(1)-离子的形成。结果清楚地表明，MALDI-MS 适用于金属标记肽的分析，这在使用 Cys 反应性 DOTA 标记的模型蛋白混合物进行 LC/MALDI 基于鉴定的蛋白中也得到了证实。与用烷基化半胱氨酸进行的运行相比，在此处鉴定到的含半胱氨酸肽增加了 50%以上。
