Single chain variable fragment displaying M13 phage library functionalized magnetic microsphere-based protein equalizer for human serum protein analysis

Guijie Zhu, Peng Zhao, Nan Deng, Dingyin Tao, Liangliang Sun, Zhen Liang, Lihua Zhang, Yukui Zhang

Research output: Contribution to journalArticle

Abstract

Single chain variable fragment (scFv) displaying the M13 phage library was covalently immobilized on magnetic microspheres and used as a protein equalizer for the treatment of human serum. First, scFv displaying M13 phage library functionalized magnetic microspheres (scFv@M13@MM) was incubated with a human serum sample. Second, captured proteins on scFv@M13@MM were eluted with 2 M NaCl, 50 mM glycine-hydrochloric acid (Gly-HCl), and 20% (v/v) acetonitrile with 0.5% (v/v) trifluoroacetic acid in sequence. Finally, the tightly bonded proteins were released by the treatment with thrombin. The eluates were first analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Results indicated that the difference of protein concentration was reduced obviously in NaCl and Gly-HCl fractions compared with untreated human serum sample. The eluates were also digested with trypsin, followed by online 2D-strong cation exchange (SCX)-RPLC-ESI-MS/MS analysis. Results demonstrated that the number of proteins identified from an scFv@M13@MM treated human serum sample was improved 100% compared with that from the untreated sample. In addition, the spectral count of 10 high abundance proteins (serum albumin, serotransferrin, α-2-macroglobulin, α-1-antitrypsin, apolipoprotein B-100, Ig γ-2 chain C region, haptoglobin, hemopexin, α-1-acid glycoprotein 1, and α-2-HS-glycoprotein) decreased evidently after scFv@M13@MM treatment. All these results demonstrate that scFv@M13@MM could efficiently remove high-abundance proteins, reduce the protein concentration difference of human serum, and result in more protein identification.

Original languageEnglish (US)
Pages (from-to)7633-7637
Number of pages5
JournalAnalytical Chemistry
Volume84
Issue number18
DOIs
StatePublished - Sep 18 2012
Externally publishedYes

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Single-Chain Antibodies
Bacteriophages
Equalizers
Microspheres
Blood Proteins
Proteins
Hydrochloric Acid
Glycine
Glycoproteins
Hemopexin
Apolipoprotein B-100
Trifluoroacetic Acid
Macroglobulins
Haptoglobins
Transferrin
Electrophoresis
Silver
Serum Albumin
Thrombin
Sodium Dodecyl Sulfate

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Single chain variable fragment displaying M13 phage library functionalized magnetic microsphere-based protein equalizer for human serum protein analysis. / Zhu, Guijie; Zhao, Peng; Deng, Nan; Tao, Dingyin; Sun, Liangliang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui.

In: Analytical Chemistry, Vol. 84, No. 18, 18.09.2012, p. 7633-7637.

Research output: Contribution to journalArticle

Zhu, Guijie ; Zhao, Peng ; Deng, Nan ; Tao, Dingyin ; Sun, Liangliang ; Liang, Zhen ; Zhang, Lihua ; Zhang, Yukui. / Single chain variable fragment displaying M13 phage library functionalized magnetic microsphere-based protein equalizer for human serum protein analysis. In: Analytical Chemistry. 2012 ; Vol. 84, No. 18. pp. 7633-7637.
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abstract = "Single chain variable fragment (scFv) displaying the M13 phage library was covalently immobilized on magnetic microspheres and used as a protein equalizer for the treatment of human serum. First, scFv displaying M13 phage library functionalized magnetic microspheres (scFv@M13@MM) was incubated with a human serum sample. Second, captured proteins on scFv@M13@MM were eluted with 2 M NaCl, 50 mM glycine-hydrochloric acid (Gly-HCl), and 20{\%} (v/v) acetonitrile with 0.5{\%} (v/v) trifluoroacetic acid in sequence. Finally, the tightly bonded proteins were released by the treatment with thrombin. The eluates were first analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Results indicated that the difference of protein concentration was reduced obviously in NaCl and Gly-HCl fractions compared with untreated human serum sample. The eluates were also digested with trypsin, followed by online 2D-strong cation exchange (SCX)-RPLC-ESI-MS/MS analysis. Results demonstrated that the number of proteins identified from an scFv@M13@MM treated human serum sample was improved 100{\%} compared with that from the untreated sample. In addition, the spectral count of 10 high abundance proteins (serum albumin, serotransferrin, α-2-macroglobulin, α-1-antitrypsin, apolipoprotein B-100, Ig γ-2 chain C region, haptoglobin, hemopexin, α-1-acid glycoprotein 1, and α-2-HS-glycoprotein) decreased evidently after scFv@M13@MM treatment. All these results demonstrate that scFv@M13@MM could efficiently remove high-abundance proteins, reduce the protein concentration difference of human serum, and result in more protein identification.",
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