Abstract
We designed the peptide microarray ChloroPhos1.0 to screen for substrates of chloroplast protein kinases. The peptides represented on the microarray were selected from phosphoproteomics data, and the identified chloroplast phosphopeptides were spotted as 15-mers on a glass slide with the phosphorylation site centered. Altogether, 905 distinct peptides from chloroplast proteins are present on the array. Here we describe how the array can be used to identify the target protein spectrum of chloroplast kinases. We present the method and discuss limitations and challenges associated with the determination of phosphorylation activity on peptide substrates in vitro.
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References
Schönberg A, Baginsky S (2012) Signal integration by chloroplast phosphorylation networks: an update. Front Plant Sci 3:256
Huang Y, Houston NL, Tovar-Mendez A et al (2010) A quantitative mass spectrometry-based approach for identifying protein kinase clients and quantifying kinase activity. Anal Biochem 402:69–76
Ubersax JA, Ferrell JE Jr (2007) Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol 8:530–541
Schönberg A, Bergner E, Helm S et al (2014) The peptide microarray “ChloroPhos1.0” identifies new phosphorylation targets of plastid casein kinase II (pCKII) in Arabidopsis thaliana. Plos One 9:108344
Frank R (1992) SPOT synthesis – an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 48:9217–9232
Wenschuh H, Volkmer‐Engert R, Schmidt M et al (2000) Coherent membrane supports for parallel microsynthesis and screening of bioactive peptides. Peptide Sci 55:188–206
Thiele A, Zerweck J, Weiwad M et al (2009) High-density peptide microarrays for reliable identification of phosphorylation sites and upstream kinases. Methods Mol Biol 570:203–219
Martin K, Steinberg TH, Goodman T (2003) Strategies and solid-phase formats for the analysis of protein and peptide phosphorylation employing a novel fluorescent phosphorylation sensor dye. Comb Chem High Throughput Screen 6:331–339
Thiele A, Weiwad M, Zerweck J et al (2010) High density peptide microarrays for proteome-wide fingerprinting of kinase activities in cell lysates. Methods Mol Biol 669:173–181
Schutkowski M, Reimer U, Panse S et al (2004) High-content peptide microarrays for deciphering kinase specificity and biology. Angew Chem Int Ed Engl 43:2671–2674
Thiele A, Stangl GI, Schutkowski M (2011) Deciphering enzyme function using peptide arrays. Mol Biotechnol 49:283–305
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Schönberg, A., Baginsky, S. (2015). The Peptide Microarray ChloroPhos1.0: A Screening Tool for the Identification of Arabidopsis thaliana Chloroplast Protein Kinase Substrates. In: Schulze, W. (eds) Plant Phosphoproteomics. Methods in Molecular Biology, vol 1306. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2648-0_11
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DOI: https://doi.org/10.1007/978-1-4939-2648-0_11
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2647-3
Online ISBN: 978-1-4939-2648-0
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