Immobilized metal affinity chromatography is a powerful method for phosphopeptide enrichment. However, the achievement of highly specific enrichment and sensitive detection of phosphopeptides by this method remains a big challenge due to the lack of high specificity and large binding capacity of conventional materials. In this work, adenosine tri-phosphate modified hydrophilic Fe₃O₄@PDA core–shell microspheres with Ti⁴⁺ exterior walls were successfully synthesized through a facile reaction at room temperature. The thicker grafting layer of the dopamine decoration exhibited enhanced hydrophilicity and biocompatibility, and the adenosine tri-phosphate molecule could offer superiorly strong and active metal phosphonate sites to bind phosphopeptides. The prepared microspheres demonstrated a low limit of detection (2 fmol), along with an exceptionally great specificity to capture phosphopeptides from a tryptic digest of the mixture of a nonphosphorylated protein BSA and a phosphorylated protein β-casein with molar ratios of BSA/β-casein up to 1000 : 1, and phosphopeptides from human serum and defatted milk were captured with high sensitivity and selectivity. In addition, the high magnetic susceptibility allowed convenient separation of the target peptides by magnetic separation. These outstanding features give the Ti⁴⁺-adenosine tri-phosphate-Fe₃O₄@polydopamine microspheres many advantages for mass spectrometric analysis of phosphopeptides.

中文翻译：

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一个新颖的IMAC平台–腺苷偶联的功能性磁性微球，用于磷酸化蛋白质组学研究†

固定化金属亲和色谱是磷酸肽富集的一种有效方法。然而，由于缺乏常规材料的高特异性和大的结合能力，通过这种方法实现磷酸肽的高特异性富集和灵敏检测仍然是一个巨大的挑战。在这项工作中，三磷酸腺苷修饰了具有Ti ^4+的亲水性Fe ₃ O ₄ @PDA核壳微球通过在室温下轻松进行反应成功地合成了外墙。多巴胺装饰物的较厚的接枝层表现出增强的亲水性和生物相容性，并且三磷酸腺苷分子可提供结合磷酸肽的强而有活性的金属膦酸酯位点。制备的微球显示出较低的检测限（2 fmol），并且具有非常高的特异性，可从非磷酸化蛋白BSA和磷酸化蛋白β-酪蛋白的摩尔比为BSA /β-的混合物的胰蛋白酶消化物中捕获磷酸肽。高达1000：1的酪蛋白，可以高灵敏度和高选择性地捕获人血清和脱脂牛奶中的磷酸肽。此外，高的磁化率允许通过磁分离方便地分离靶肽。这些出色的功能使Ti⁴⁺腺苷三磷酸-Fe ₃ O ₄聚多巴胺微球在质谱分析磷酸肽方面具有许多优势。