Ole Pless, PhD;
Fraunhofer IME Screening Port, Hamburg, Germany
“We used the protein macroarrays to screen for protein-protein interaction (PPI) partners using fluorophore-labelled peptide baits. This enabled us to identify the histone lysine Nmethyltransferase EHMT2/G9a as an interaction partner and functional modulator of trans-activation domains of transcription factors of the C/EBP family1. We have further extended these PPI screens on UniPEx Arrays and to differentially post-translationally modified (PTM) peptides as baits. Thereby, we could not only measure large numbers of protein interactions on approximately one-third of the proteome simultaneously, but also explore a protein´s interactome depending on defined PTM states2. An advantage of this approach using UniPEx protein/peptide libraries was the presence of large amounts of unmodified pray protein (expressed in E. coli) in combination with a defined duplicate spotting pattern, which enables quick exclusion of false-positive interactions.”
1 Pless O, Kowenz-Leutz E, Knoblich M, Lausen J, Beyermann M, Walsh MJ & Leutz A.(2008), G9a-mediated Lysine Methylation Alters the Function of CCAAT/Enhancer-binding Protein-beta. The Journal of Biological Chemistry VOL. 283, NO. 39, pp. 26357–26363; DOI 10.1074/jbc.M8021322002.
2 Pless O, Kowenz-Leutz E, Dittmar G & Leutz A (2011), A differential proteome screening system for post-translational modification–dependent transcription factor interactions. Nature Protocols VOL.6 NO. 3; doi:10.1038/nprot.2011.303
Şükrü Atakan, Postdoctoral Researcher;
Bilkent University, Turkey
“To our experience, the protein arrays are super-informative and yield highly reproducible results specifically for biomarker discovery and validation. We have screened Testis, Fetal hEX1, UniPEx and hEXselect protein arrays to identify autologous antibodies specific to Small Cell Lung Cancer (SCLC), Non-small Cell Lung Cancer (NSLCL), Ovarian Cancer, Colorectal Cancer, Gastric Cancer, Behcet's disease and Neuromyelitis optica (NMO) in comparison with healthy controls. We were able to identify disease specific autologous antibodies and validate them via custom-made arrays.
The nature and format of the protein arrays provided us the opportunity to develop a screening protocol by which we were able to generate signals in a broad dynamic range, obtain high signal/background and positive signal/negative signal ratios. Further, we could identify novel autologous antibodies which we will publish after validation with ELISA and Western blot”
Bernhard Reuss, PhD; Professor for Cellular Neuroanatomy,
University Medical Center Göttingen
„We used the hEXselect multiprotein-array to identify interaction partners of antisera directed to bacteria, which are suspected to cause schizophrenia in the offspring of mothers infected during early pregnancy.1,2 By this we could identify a handful of interesting candidate molecules which have later been functionally characterized. In the course of these experiments, the hEXselect multiprotein-array turned out to be a highly valuable tool that is comparably easy to handle, making the experimental procedures nearly as simple as a western blot!
However, even more important for our project was the fact, that the cDNAs in the expression library used for this array, were derived from the frontal cortex of two human fetal brains of the late first and the early second trimester of pregnancy, thereby covering in terms of schizophrenia pathology almost perfectly the expression pattern during the most vulnerable phase of prenatal brain development.“
1 Almamy A, Schwerk C, Schroten H, Ishikawa H, Asif AR, Reuss B. 2017. Crossreactivity of an Antiserum Directed to the Gram-Negative Bacterium Neisseria gonorrhoeae with the SNARE-Complex Protein Snap23 Correlates to Impaired Exocytosis in SH-SY5Y Cells. J Mol Neurosci 62(2):163-180.
2 Almamy A, Schwerk C, Schroten H, Ishikawa H, Asif AR, Reuss B. 2017. Interactions of antisera to different Chlamydia and Chlamydophila species with the ribosomal protein RPS27a correlate with impaired protein synthesis in a human choroid plexus papilloma cell line. Immunol Res 65:1110-1123.