Post-translational modification of proteins by phosphorylation and dephosphorylation is a common mechanism used for regulating signal transduction cascades and cellular physiology. Protein kinases catalyze phosphorylation of serine, threonine and tyrosine residues on proteins while phosphatases mediate removal of phosphate groups from these residues.  Deregulation of phosphatases has been implicated in a variety of diseases (e.g., cancer, diabetes, cardiac hypertrophy, and neurodegeneration). Currently there is a lack of a universal technique suitable for analyzing activity of protein phosphatases in a complex sample such as cell lysate and biofluid.<br/>      This presentation describes combination of peptide arrays and matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) for high throughput assays of phosphatase activities in lysates of cultured melanocytes and cancerous C8161 melanoma cells. More than 700 phosphopeptides were prepared through combinatorial synthesis, and immobilized to self-assembled monolayers (SAMs) on 384-spot plates. The peptide sequences and immobilization chemistry were thoughtfully designed to ensure the control over enzyme-substrate interaction, sensitivity and quantitation of MS detection. MALDI MS data was generated through automatic plate scanning and processed by customized computer software to determine the degree of dephosphorylation of each peptide on the arrays, and to reveal patterns of phosphatase activities. Peptide arrays treated with cancerous melanoma cell lysates demonstrated significantly increased dephosphorylation of phosphopeptides compared to those treated with noncancerous melanocyte lysate. In addition by using selective phosphatase inhibitors we were able to distinguish the contribution from serine/threonine phosphatases, tyrosine phosphatases and dual-specific phosphatases in these cell lysates.<br/>       This work establishes a platform to decipher protein phosphatase activity in a high throughput manner. The use of a label-free analytical method makes this technique readily applicable to a broader range of enzyme activities and provides a new opportunity for identifying changes in global activity profiles that characterize the states of cells.

Profiling protein phosphatase activity using peptide arrays and mass spectrometry

251st National Meeting (2016)

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