Citation: DONG Li-Jun, ZHANG Li-Yuan, PENG Jin-Yong, DONG Pei-Pei, WANG Li-Li, ZHANG Li-Juan, HOU Xiao-Lin, ZHAO Yan-Yan. Application of Novel Reversed Phase/Strong Anion Exchange Material in Enrichment and Fractionation of Phosphopeptides[J]. Chinese Journal of Analytical Chemistry, ;2017, 45(1): 61-68. doi: 10.11895/j.issn.0253-3820.160415 shu

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

1.
Dalian Medical University, Pharmacy College, Dalian 116044, China;
2.
Songyuan City Qianguo Traditional Chinese Medical Hospital of Jilin Province, Songyuan 131100, China;
3.
Dalian Medical University, College of Basic Medicine, Dalian 116044, China

Received Date: 31 May 2016
Revised Date: 12 November 2016

Fund Project: This work was supported by the National Natural Science Foundation of China (Nos.21105007,21505015,21475129)

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 (C₁₈/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 α-casein digests as model sample. It was found that the retention of phosphopeptides on C₁₈/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 C₁₈/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 α-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.
- Phosphopeptides,
- Reversed phase/strong anion exchange material,
- Enrichment
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