二氧化钛富集法作为目前使用最为广泛的金属氧化物富集磷酸肽的方法,在富集过程中常常对富含天冬氨酸和谷氨酸的酸性非磷酸化肽段存在一定的非特异性吸附作用.这些肽段与磷酸化肽段一同被富集,降低了磷酸肽富集的选择性.传统方法中使用的非特异性吸附抑制剂常会对质谱的电喷雾离子源造成污染,因而限制了其在液相色谱-质谱联用(LC-MS)系统中的应用.本研究将天冬氨酸作为一种新型的非特异性吸附抑制剂加入到二氧化钛富集体系中,并分别对3种和9种标准蛋白质酶切肽段混合物进行富集实验,同时与添加另一种非特异性吸附抑制剂--谷氨酸以及不添加任何非特异性吸附抑制剂的富集体系进行了富集效果的比较.结果表明,天冬氨酸可以有效地提高二氧化钛对磷酸肽富集的选择性.将添加天冬氨酸的二氧化钛富集体系应用于鼠肝全蛋白质磷酸肽的富集中,同样取得了很好的效果,表明天冬氨酸在复杂的生物样本的磷酸肽富集中也同样具有良好的应用前景.此外,由于天冬氨酸在反相色谱中极易被洗脱去除,从而避免了传统抑制剂对LC-MS系统离子源的污染问题.
Titanium dioxide (TiO_2) is one of metal oxides widely used for phosphopeptide enrichment in phosphoproteomic research nowadays.However it can bind to some non-phosphorylated peptides containing one or more aspartic acid residues and/or glutamic acid residues.These non-phosphorylated peptides can be eluted along with phosphorylated peptides and cause the reduction of the selectivity.Conventional inhibitors for the non-specific binding of non-phosphorylated peptides can often contaminate the ion source of mass spectrometry and therefore their applications are limited in liquid chromatography-mass spectrometry (LC-MS).In this study,aspartic acid was reported as a novel non-specific binding inhibitor for phosphopeptide enrichment by titanium dioxide.Firstly,the tryptic peptide mixtures of 3 and 9 standard proteins were used for the comparison of the enrichment efficiency of titanium dioxide.The effects with the presence of aspartic acid,glutamic acid and no-inhibitor in the enrichment systems were compared separately.The results showed that aspartic acid can greatly improve the selectivity of titanium dioxide for phosphopeptide enrichment.Then,aspartic acid was used for the enrichment of tryptic peptide mixture of C57BL/6J mouse liver lysate and good results were also obtained which demonstrated that aspartic acid was a promising non-specific binding inhibitor for complex biological samples.Besides,no contamination in the ion source occurred during the mass spectrometric analysis.
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