Single chain variable fragment displaying M13 phage library functionalized magnetic microsphere-based protein equalizer for human serum protein analysis.
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.
Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R. and A. Center, Dalian Institute of Chemical Physics, The Chinese Academy of Science, Dalian, China.
Guijie
G
Zhu
Peng
P
Zhao
Nan
N
Deng
Dingyin
D
Tao
Liangliang
L
Sun
Zhen
Z
Liang
Lihua
L
Zhang
Yukui
Y
Zhang
D055910:Antibodies, Immobilized / Q000737:chemistry / Q000378:metabolism; D017104:Bacteriophage M13 / Q000378:metabolism*; D001798:Blood Proteins / Q000032:analysis*; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008280:Magnetics; D008863:Microspheres; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D055910:Antibodies, Immobilized; D001798:Blood Proteins; D019151:Peptide Library; D057127:Single-Chain Antibodies; D014357:Trypsin; D013917:Thrombin