Novel Ser/Thr protein phosphatase 5 (PP5) regulated targets during DNA damage identified by proteomics analysis.

The DNA damage response likely includes a global phosphorylation signaling cascade process for sensing the damaged DNA condition and coordinating responses to cope with and repair the perturbed cellular state. We utilized a label-free liquid chromatography-mass spectrometry approach to evaluate changes in protein phosphorylation associated with PP5 activity during the DNA damage response. Biological replicate analyses of bleomycin-treated HeLa cells expressing either WT-PP5 or mutant inactive PP5 lead to the identification of six potential target proteins of PP5 action.

The influence of sample preparation and replicate analyses on HeLa Cell phosphoproteome coverage.

Ongoing optimization of proteomic methodologies seeks to improve both the coverage and confidence of protein identifications. The optimization of sample preparation, inclusion of technical replicates (repeated instrumental analysis of the same sample), and biological replicates (multiple individual samples) are crucial in proteomic studies to avoid the pitfalls associated with single point analysis and under-sampling. Phosphopeptides were isolated from HeLa cells and analyzed by nano-reversed phase liquid chromatography electrospray ionization tandem mass spectrometry (nano-RP-LC-MS/MS).

Applying a targeted label-free approach using LC-MS AMT tags to evaluate changes in protein phosphorylation following phosphatase inhibition.

To identify phosphoproteins regulated by the phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of changes in protein phosphorylation on extracts from HeLa cells treated with or without calyculin A, a potent PPP enzyme inhibitor. A label-free comparative phosphoproteomics approach using immobilized metal ion affinity chromatography and targeted tandem mass spectrometry was employed to discover and identify signatures based upon distinctive changes in abundance. Overall, 232 proteins were identified as either direct or indirect targets for PPP enzyme regulation.

Cellular co-localization of protein phosphatase 5 and glucocorticoid receptors in rat brain.

Glucocorticoid receptors are widely expressed in brain, where they are thought to play a role in controlling neurogenesis and to mediate many of the central nervous system effects of stress. In non-neuronal cells, protein phosphatase 5 (PP5) has been found in complexes with heat shock protein 90 and glucocorticoid receptors and may be a negative modulator of glucocorticoid receptor function. In the present study, we used co-immunofluorescence analysis to examine whether PP5 and glucocorticoid receptors are co-expressed at the cellular level in rat brain.