title: Improved intra-array and interarray normalization of peptide microarray phosphorylation for phosphorylome and kinome profiling by rational selection of relevant spots.
abstract: Massive parallel analysis using array technology has become the mainstay for analysis of genomes and transcriptomes. Analogously, the predominance of phosphorylation as a regulator of cellular metabolism has fostered the development of peptide arrays of kinase consensus substrates that allow the charting of cellular phosphorylation events (often called kinome profiling). However, whereas the bioinformatical framework for expression array analysis is well-developed, no advanced analysis tools are yet available for kinome profiling. Especially intra-array and interarray normalization of peptide array phosphorylation remain problematic, due to the absence of "housekeeping" kinases and the obvious fallacy of the assumption that different experimental conditions should exhibit equal amounts of kinase activity. Here we describe the development of analysis tools that reliably quantify phosphorylation of peptide arrays and that allow normalization of the signals obtained. We provide a method for intraslide gradient correction and spot quality control. We describe a novel interarray normalization procedure, named repetitive signal enhancement, RSE, which provides a mathematical approach to limit the false negative results occuring with the use of other normalization procedures. Using in silico and biological experiments we show that employing such protocols yields superior insight into cellular physiology as compared to classical analysis tools for kinome profiling.
authors.affiliation: Department of Developmental BioEngineering, MIRA institute for biomedical technology and technical medicine, University of Twente, P.O. Box 217, NL-7500 AE Enschede, The Netherlands.
authors.forename: Jetse
authors.initials: J
authors.lastname: Scholma
authors.affiliation: Department of Gastroenterology and Hepatology. Erasmus MC, University Medical Center Rotterdam, 's Gravendijkwal 230, NL-3015 CE Rotterdam, The Netherlands.
authors.forename: Gwenny M
authors.initials: GM
authors.lastname: Fuhler
authors.affiliation: Pepscope BV, Dantelaan 83, 3533 VB Utrecht, The Netherlands.
authors.forename: Jos
authors.initials: J
authors.lastname: Joore
authors.affiliation: Department of Clinical Genetics, VU University Medical Center, 1007 MB Amsterdam, The Netherlands.|Delft Bioinformatics Lab. Delft University of Technology, Mekelweg 4, NL-2628 CD Delft, The Netherlands.
authors.forename: Marc
authors.initials: M
authors.lastname: Hulsman
authors.affiliation: Department of Formal Methods and Tools, CTIT institute, University of Twente, P.O. Box 217, NL-7500 AE Enschede, The Netherlands.
authors.forename: Stefano
authors.initials: S
authors.lastname: Schivo
authors.affiliation: Department of Malignant Hematology, Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA.
authors.forename: Alan F
authors.initials: AF
authors.lastname: List
authors.affiliation: Delft Bioinformatics Lab. Delft University of Technology, Mekelweg 4, NL-2628 CD Delft, The Netherlands.
authors.forename: Marcel J T
authors.initials: MJ
authors.lastname: Reinders
authors.affiliation: Department of Gastroenterology and Hepatology. Erasmus MC, University Medical Center Rotterdam, 's Gravendijkwal 230, NL-3015 CE Rotterdam, The Netherlands.
authors.forename: Maikel P
authors.initials: MP
authors.lastname: Peppelenbosch
authors.affiliation: Department of Developmental BioEngineering, MIRA institute for biomedical technology and technical medicine, University of Twente, P.O. Box 217, NL-7500 AE Enschede, The Netherlands.
authors.forename: Janine N
authors.initials: JN
authors.lastname: Post
mesh_terms: D005260:Female; D006801:Humans; D008297:Male; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*
chemical_list: D010750:Phosphoproteins