High-throughput workflow for identification of phosphorylated peptides by LC-MALDI-TOF/TOF-MS coupled to in situ enrichment on MALDI plates functionalized by ion landing.

We report an MS-based workflow for identification of phosphorylated peptides from trypsinized protein mixtures and cell lysates that is suitable for high-throughput sample analysis. The workflow is based on an in situ enrichment on matrix-assisted laser desorption/ionization (MALDI) plates that were functionalized by TiO2 using automated ion landing apparatus that can operate unsupervised. The MALDI plate can be functionalized by TiO2 into any array of predefined geometry (here, 96 positions for samples and 24 for mass calibration standards) made compatible with a standard MALDI spotter and coupled with high-performance liquid chromatography. The in situ MALDI plate enrichment was compared with a standard precolumn-based separation and achieved comparable or better results than the standard method. The performance of this new workflow was demonstrated on a model mixture of proteins as well as on Jurkat cells lysates. The method showed improved signal-to-noise ratio in a single MS spectrum, which resulted in better identification by MS/MS and a subsequent database search. Using the workflow, we also found specific phosphorylations in Jurkat cells that were nonspecifically activated by phorbol 12-myristate 13-acetate. These phosphorylations concerned the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and its targets and were in agreement with the current knowledge of this signaling cascade. Control sample of non-activated cells was devoid of these phosphorylations. Overall, the presented analytical workflow is able to detect dynamic phosphorylation events in minimally processed mammalian cells while using only a short high-performance liquid chromatography gradient.