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High-performance thin-layer chromatography as a fast screening tool for phosphorylated peptides

: Morschheuser, Lena; Mükusch, Sandra; Riedner, Maria; Seitz, Harald; Rohn, Sascha


Journal of chromatography. B 1008 (2016), pp.198-205
ISSN: 0378-4347
ISSN: 1570-0232
Journal Article
Fraunhofer IZI ()
high-performance thin-layer chromatography; phosphopeptide detection; nociceptive pathway; HPTLC-immunostaining

This study aimed at developing a rapid chromatographic assay to monitor phosphorylation sites in peptides. For the analysis of nociceptive signal transduction pathways, the detection of phosphorylated proteins/peptides plays a fundamental role. To get further insights in the phosphorylation mechanism of protein kinase C-ε (PKC-ε) and protein kinase A (PKA), potential targets were divided into subsections resulting in peptides that contain only one possible phospho-binding site. The use of high-performance thin-layer chromatography (HPTLC) offers the possibility of a high throughput of samples and the advantage of a quick sample clean-up. A combined strategy of an effect-directed overlay procedure on the TLC plate using specific antibodies (immunostaining, HPTLC-IS) as well as a parallel, direct mass spectrometric methodology by HPTLC-MALDI-TOF-MS was developed. With regard to HPTLC-IS, validation of the data exhibited a lower limit of detection than the traditionally used protein derivatization reagent fluorescamine. Besides the identification of the phosphorylated peptides, a semi-quantitative estimation can be performed with HPTLC-IS.