亲水模式下流动相pH值对磷酸化肽在Click OEG-CD材料上富集选择性的影响

以标准蛋白质α-酪蛋白的酶解液作为研究对象,考察流动相pH值对磷酸化肽在Click OEG-CD材料上富集选择性的影响。首先以磷酸苯二钠作为模型化合物考察流动相pH值对其在Click OEG-CD材料上的保留影响,结果表明当pH值低于磷酸根的pK_a值时,磷酸苯二钠难以电离,与材料的离子交换作用较弱,因而保留也较弱。然后在亲水模式下流动相pH值分别为2, 4, 6时考察Click OEG-CD材料对α-酪蛋白的酶解液中磷酸化肽的富集选择性影响。结果表明,当流动相pH为2时,磷酸化肽不能被材料富集;当pH为4时,磷酸化肽能够被富集,而且洗脱窗口较窄;当pH为6时,磷酸化肽也能够被富集,但是洗脱窗口较宽。因此适合亲水模式下富集磷酸化肽的流动相pH值为4。本研究结果能够为今后将Click OEG-CD材料更好的应用于磷酸化肽富集提供有意义的借鉴。

Influence of pH value of mobile phase on phosphopeptide enrichment selectivity under hydrophilic interaction liquid chromatography mode by using Click OEG-CD matrix

The digest of the standard proteinα-casein was used as the objective to investigate the influence of pH value of mobile phase on phosphopeptide enrichment selectivity by using Click OEG-CD matrix. Disodium phenyl phosphate was primarily selected as the model sample. The results indicated that phosphate group was difficultly to be ionized when the pH value of mobile phase was below its pK_a value. The retention of disodium phenyl phosphate on the Click OEG-CD matrix was not based on ion exchange interaction, leading to the weak retention. The influence of pH value (2, 4 and 6) of mobile phase under hydrophilic interaction liquid chromatography (HILIC) mode on phosphopeptide enrichment selectivity was investigated taking the digest of α-casein as a sample. Phosphopeptides could not be enriched on the matrix with the pH value of 2; phosphopeptides could be enriched on the matrix and the elution window was narrow with the pH value of 4; phosphopeptides could be enriched on the matrix while the elution window was broad with the pH value of 6. According to the investigation, Click OEG-CD matrix could be better applied in phosphopeptide enrichment.