Urinary proteome, as an important component of body fluid proteome, could reflect kidney, urogenital tract function and pathological changes of human organs. This study reports a convenient strategy for urine proteome analysis through ampholine immobilized polymer microsphere (ampholine@PM) fractionation strategy. After ampholine@PM treatment, 16,543 unique peptides corresponding to 2173 non-redundant urinary proteins were identified, while only 856 proteins, corresponding to 3524 peptides were identified in the crude urine sample. The number of proteins and peptides was increased by 1.54 and 3.69 times, respectively. 31 urinary candidate biomarkers have also been identified (17 candidate biomarkers of glomerular injury and 14 candidate biomarkers of tubular injury), showing the potential of our strategy in urinary biomarker discovery study. In additional to the urine proteome, N-glycoproteome analysis was also performed after ampholine@PM fractionation followed by the N-glycopeptides enrichment. The number was increased from 144 to 281 for N-glycoproteins, 261 to 709 for N-glycopeptides, and 226 to 493 for N-glycosylation sites, after ampholine@PM treatment. Based on the significant increase on the identified N-glycoprotein number, ampholine@PM fractionation strategy also offered a beneficial tool for the post translational modification analysis of urine proteome.

尿蛋白质组作为体液蛋白质组的重要组成部分，可以反映肾脏，泌尿生殖道的功能和人体器官的病理变化。这项研究报告了通过固定两性霉素的聚合物微球（ampholine @ PM）分级策略进行尿液蛋白质组分析的便捷策略。经过两性霉素@PM处理后，在粗尿液样品中鉴定出了16,543个独特肽，对应于2173种非冗余尿蛋白，而仅鉴定了856个蛋白，对应于3524个肽。蛋白质和肽的数量分别增加了1.54和3.69倍。还确定了31种尿液候选生物标志物（肾小球损伤的17种候选生物标志物和肾小管损伤的14种候选生物标志物），显示了我们在尿液生物标志物发现研究中的策略潜力。除尿蛋白组蛋白外，还在两性霉素@PM分离后富集N-糖肽后还进行了N-糖蛋白组分析。经两性霉素@PM处理后，N-糖蛋白的数量从144增加到281，N-糖肽从261增加到709，N-糖基化位点从226增加到493。基于已鉴定的N-糖蛋白数量的显着增加，ampholine @ PM分级分离策略也为尿蛋白组的翻译后修饰分析提供了有益的工具。