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建立了新型反相/强阴离子交换混合模式材料(C18/SAX)的磷酸化肽富集方法。考察了流动相组成(乙腈浓度、甲酸浓度、缓冲盐浓度)对酪蛋白(α-Casein)酶解液中磷酸化肽分离选择性的影响。实验结果表明,磷酸化肽在C18/SAX上的保留行为受疏水和离子交换作用力的共同调控,单磷酸化肽先于多磷酸化肽从材料上洗脱出来。随着甲酸浓度增加,磷酸化肽的保留减弱;随着盐浓度增加,磷酸化肽保留变小。采用优化后的流动相,建立以20%ACN/20 mmol/L NH4Ac作为上样溶液,20%ACN/0.1%FA和50%ACN/100 mmol/L NH4Ac/2%FA分别作为洗脱液分段洗脱单、多磷酸化肽的方法。以α-Casein和人血清白蛋白(HSA)酶解液的混合溶液(1∶20,n/n)作为模拟样品,实现了单、多磷酸化肽的同时富集和分段洗脱,分别检测到4条单磷酸化肽和14条多磷酸化肽的信号。将本方法用于牛奶中的磷酸化肽检测,共鉴定到4条单磷酸化肽和8条多磷酸化肽信号。结果表明,本富集方法选择性高,有良好的应用前景。
A novel method of phosphopeptide enrichment of reversed-phase / strong anion exchange hybrid mode materials (C18 / SAX) was established. The effect of mobile phase composition (acetonitrile concentration, formic acid concentration and buffer salt concentration) on the selectivity of phosphopeptides in the casein (α-Casein) hydrolyzate was investigated. The experimental results show that the retention of phosphopeptides on C18 / SAX is co-regulated by both hydrophobic and ion exchange forces, and the monophosphorylated peptide elutes from the material prior to the polyphosphorylated peptide. As the formic acid concentration increases, the retention of phosphopeptides diminishes; as the salt concentration increases, the phosphopeptide remains less retained. Using the optimized mobile phase, 20% ACN / 20 mmol / L NH4Ac as the loading solution, 20% ACN / 0.1% FA and 50% ACN / 100 mmol / L NH4Ac / 2% Single-stage elution, polyphosphorylation of peptides. The mixed solution of α-Casein and human serum albumin (HSA) hydrolyzate (1:20, n / n) was used as a sample to achieve the simultaneous enrichment and elution of single and polyphosphorylated peptides respectively Four monophosphorylated peptides and 14 polyphosphorylated peptides were detected. The method was applied to the detection of phosphopeptides in milk. Four monophosphorylated peptides and eight polyphosphorylated peptide signals were identified. The results show that the enrichment method has high selectivity and good application prospect.