Antibodies Immobilized as Arrays to Profile Protein Post-translational Modifications in Mammalian Cells

Abstract

Previously, we demonstrated that antibodies printed on a solid support were able to detect protein-protein interaction in mammalian cells. Here we further developed the antibody array system for detecting proteins with various post-translational modifications in mammalian cells. In this novel approach, immunoprecipitated proteins were labeled with fluorescent dye followed by incubation over antibody arrays. Targeted proteins, captured by the antibodies immobilized on PVDF membrane or glass slide, were detected by means of near infrared fluorescent scanner or fluorescent microscopy. To demonstrate the application of the antibody arrays in protein post-translational modifications, we profiled protein tyrosine phosphorylation, ubiquitination, and acetylation in mammalian cells under different conditions. Our results indicate that antibody array technology can provide a powerful means of profiling a large number of proteins with different post-translational modifications in cells. Previously, we demonstrated that antibodies printed on a solid support were able to detect protein-protein interaction in mammalian cells. Here we further developed the antibody array system for detecting proteins with various post-translational modifications in mammalian cells. In this novel approach, immunoprecipitated proteins were labeled with fluorescent dye followed by incubation over antibody arrays. Targeted proteins, captured by the antibodies immobilized on PVDF membrane or glass slide, were detected by means of near infrared fluorescent scanner or fluorescent microscopy. To demonstrate the application of the antibody arrays in protein post-translational modifications, we profiled protein tyrosine phosphorylation, ubiquitination, and acetylation in mammalian cells under different conditions. Our results indicate that antibody array technology can provide a powerful means of profiling a large number of proteins with different post-translational modifications in cells. Protein function and half-life are often under the tight regulation of post-translational modifications such as phosphorylation, ubiquitination, and acetylation. Tyrosine phosphorylation is a key mechanism for the reversible regulation of protein activity during signal transduction. The polyubiquitination-proteosome pathway plays a substantial role in the degradation of regulatory proteins in a variety of cellular processes. As for acetylation, accumulating evidence indicates that transcriptional regulation is strongly influenced by the acetylation of histones as well as transcription factors. For many proteins, multiple post-translational modifications may occur simultaneously or sequentially. In the case of p53 tumor suppressor, ubiquitination, phosphorylation, and acetylation can all affect its activity (1Brooks C.L. Gu W. Ubiquitination, phosphorylation and acetylation: The molecular basis for p53 regulation..Curr. Opin. Cell Biol. 2003; 15: 164-171Google Scholar). Altered protein tyrosine phosphorylation, protein ubiquitination, and protein acetylation in mammalian cells are closely related to developmental diseases and cancer. Recently, mass spectrometry has been employed for identification of proteins or peptides or more specifically, the residues bearing phosphorylation/acetylation modifications (2Wang Y.H. Tsay Y.G. Tan B.C. Lo W.Y. Le S.C. Identification and characterization of a novel p300-mediated p53 acetylation site, lysine 305..J. Biol. Chem. 2003; 278: 25568-25576Google Scholar, 3Shi Y. Sawada J. Sui G. Affar el B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Shi Y. Coordinated histone modifications mediated by a CtBP co-repressor complex..Nature. 2003; 422: 735-738Google Scholar). However, a convenient and reliable biochemical method for identifying proteins with the aforementioned post-translational modifications as they occur within a cell lysate is still lacking. We recently immobilized 50–100 different antibodies on a PVDF membrane for identification of