A novel protein equalizer based on single chain variable fragment display M13 phage library for nephropathy patient urine study

Peng Zhao, Dingyin Tao, Zhen Liang, Lihua Zhang, Yukui Zhang

Research output: Contribution to journalArticlepeer-review

Abstract

A novel protein equalizer was developed with single chain variable fragment (scFv) library displaying M13 phage covalently bonded on monolithic cryogel. Due to the great number and various kinds of displayed scFv fragments, as well as strong and specific binding capacity between scFv fragments and proteins, a new protein equalizer technology is preferable in the pretreatment of complex protein samples. After the sample dissolved in phosphate buffer solution (PBS), it was repeatedly loaded onto the equalizer for five times, the bound proteins were in sequence eluted by 2 mol/L NaCl and 50 mmol/L Gly-HCl (pH 2.5) solution, followed by digestion with thrombin. All proteins or peptides collected from each fraction were further analyzed by high performance liquid chromatography-electrospray tandem mass spectrometry (RPLC-ESI-MS/MS) with a serially coupled long microcolumn. Compared with the untreated samples, the identified protein number was increased from 142 to 396. Furthermore, from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis results, it was found that the protein concentration difference was reduced obviously in the eluant of direct sample loading, and most high abundance proteins were identified in the eluant of NaCl. All these results demonstrate that the novel protein equalizer with scFv display M13 phage library immobilized on cyrogel could effectively reduce the dynamic range of proteins in complex samples, enabling the identification of more low abundance proteins.

Original languageEnglish (US)
Pages (from-to)249-253
Number of pages5
JournalChinese Journal of Chromatography (Se Pu)
Volume27
Issue number3
StatePublished - May 1 2009

Keywords

  • Liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) with a serially coupled long microcolumn
  • Nephropathy patient
  • Protein
  • Protein equalizer
  • Single chain variable fragment (scFv) display M13 phage library
  • Urine

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Chemical Engineering(all)
  • Organic Chemistry
  • Electrochemistry

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