Proteomics analysis of co-purifying cellular proteins associated with rAAV vectors.
Recombinant adeno-associated vectors (rAAV) are commonly purified by either chromatography or equilibrium CsCl gradient. Nevertheless, even after purification various cellular proteins often associate with rAAV vector capsids. Such co-purifying cellular proteins may raise concern about safety of gene therapy. Here we report identification and characterization of the co-purifying cellular protein in the vector preparations by using a combination of two proteomics approaches, GeLC-MS (gel electrophoresis liquid chromatography-mass spectrometry) and 2DE (two-dimensional gel electrophoresis). Most prominent bands revealed by Coomassie Blue staining were mostly similar to the AAV capsid proteins. Posttranslational modifications of capsid proteins were detected by the proteomics analysis. A total of 13 cellular proteins were identified in the rAAV vectors purified by two rounds of cesium chloride gradient centrifugation, including 9 by the GeLC-MS analysis and 4 by the 2DE analysis. Selected cellular proteins were verified by western blot. Furthermore, the cellular proteins could be consistently found associated with different AAV serotypes and carrying different transgenes. Yet, the proteins were not integral components of the viral capsis since a stringent washing procedure by column purification could remove them. These co-purified proteins in AAV vector preparations may have a role in various stages of the AAV life cycle.
Department of Microbiology and Immunology, Sol Sherry Thrombosis Research Center, Temple University, Philadelphia, Pennsylvania, United States of America.
Biao
B
Dong
Department of Microbiology and Immunology, Sol Sherry Thrombosis Research Center, Temple University, Philadelphia, Pennsylvania, United States of America.
Xunbao
X
Duan
Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.
Hoi Yee
HY
Chow
Department of Microbiology and Immunology, Sol Sherry Thrombosis Research Center, Temple University, Philadelphia, Pennsylvania, United States of America.
Lingxia
L
Chen
Department of Microbiology and Immunology, Sol Sherry Thrombosis Research Center, Temple University, Philadelphia, Pennsylvania, United States of America.
Hui
H
Lu
Department of Microbiology and Immunology, Sol Sherry Thrombosis Research Center, Temple University, Philadelphia, Pennsylvania, United States of America.
Wenman
W
Wu
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, and Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America.
Bernd
B
Hauck
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, and Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America.
Fraser
F
Wright
St. Laurent Institute, Cambridge, Massachusetts, United States of America.
Philipp
P
Kapranov
Department of Microbiology and Immunology, Sol Sherry Thrombosis Research Center, Temple University, Philadelphia, Pennsylvania, United States of America.
Weidong
W
Xiao
D015153:Blotting, Western; D036022:Capsid Proteins / Q000032:analysis* / Q000302:isolation & purification; D002853:Chromatography, Liquid; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D015316:Genetic Therapy / Q000379:methods / Q000592:standards; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D036022:Capsid Proteins; D020543:Proteome