Application of Novel Reversed Phase/Strong Anion Exchange Material in Enrichment and Fractionation of Phosphopeptides

作者：Dong Li-Jun; Zhang Li-Yuan; Peng Jin-Yong; Dong Pei-Pei; Wang Li-Li; Zhang Li-Juan; Hou Xiao-Lin; Zhao Yan-Yan^*

来源：Chinese Journal of Analytical Chemistry, 2017, 45(1): 61-68.

DOI：10.11895/j.issn.0253.3820.160415

• Phosphopeptides
• Reversed phase/strong anion exchange material
• Enrichment

Selective enrichment before mass spectrometric analysis is very important for detection and identification of phosphopeptides. In this work, a novel method based on the reversed phase/strong anion exchange material (C-18/SAX) was established and applied in phosphopeptide enrichment. The influence of mobile phase composition (acetonitrile content, formic acid content, salt concentration) on the phosphopeptide enrichment selectivity was carried out by taking alpha-casein digests as model sample. It was found that the retention of phosphopeptides on C-18/SAX was ascribed to both the hydrophobic and strong anion exchange interactions. Mono-phosphopeptides were eluted earlier than multi-phosphopeptides. The retention of phosphopeptides on C-18/SAX was reduced along with the increase of formic acid content and the increase of salt content. After optimization, an enrichment method was developed with 20% ACN/20 mmol/L NH4Ac as loading buffer, 20% ACN/0. 1% FA and 50% ACN/100 mmol/L NH4Ac/2% FA as two steps elution phase to fractionate mono-and multi-phosphopeptides. By using the developed method, 4 mono-phosphopeptides and 14 multi-phosphopeptides were isolated from the mixture of alpha-Casein and HSA at a molar ratio of 1: 20. The application of the developed method to bovine milk digests resulted in the separation and identification of 4 mono-phosphopeptide and 8 multi-phosphopeptides. These results indicated that the methods had great potential for practical application.

• 出版日期2017-1
• 单位大连医科大学