Staphylococcus aureus is a major cause of infections worldwide, and infection results in a variety of diseases. As of no surprise, protein phosphorylation is an important game player in signaling cascades and has been shown to be involved in S. aureus virulence. Albeit long neglected, eukaryotic-type serine/threonine kinases in S. aureus have been implicated in this complex signaling cascades. Due to the substoichiometric nature of protein phosphorylation and a lack of suitable analysis tools, the knowledge of these cascades is, however, to date, still limited. Here, were apply an optimized protocol for efficient phosphopeptide enrichment via Fe 3+ -IMAC followed by LC-MS/MS to get a better understanding of the impact of protein phosphorylation on the complex signaling networks involved in pathogenicity. By profiling a serine/threonine kinase and phosphatase mutant from a methicillin-resistant S. aureus mutant library, we generated the most comprehensive phosphoproteome data set of S. aureus to date, aiding a better understanding of signaling in bacteria. With the identification of 3800 class I p-sites, we were able to increase the number of identifications by more than 21 times compared with recent literature. In addition, we were able to identify 74 downstream targets of the only reported eukaryotic-type Ser/Thr kinase of the S. aureus strain USA300, Stk1. This work allowed an extensive analysis of the bacterial phosphoproteome and indicates that Ser/Thr kinase signaling is far more abundant than previously anticipated in S. aureus .

Recent technological advances have made it possible to investigate the hitherto rather elusive protein histidine phosphorylation. However, confident site-specific localization of protein histidine phosphorylation remains challenging. Here, we address this problem, presenting a mass-spectrometry-based approach that outperforms classical HCD fragmentation without compromising sensitivity. We use the phosphohistidine immonium ion as a diagnostic tool as well as ETD-based fragmentation techniques to achieve unambiguous identification and localization of histidine-phosphorylation sites. The work presented here will allow more confident investigation of the phosphohistidine proteome to reveal the roles of histidine phosphorylation in cellular signaling events.

Targeted therapies against oncogenic receptor tyrosine kinases (RTK) show promising results in the clinic. Unfortunately, despite the initial positive response, most patients develop therapeutic resistance. Most research has focused on acquired resistance occurring after an extensive time of treatment; however, the question remains as to how cells can survive an initial treatment, as early resistance to apoptosis will enable cells to develop any growth-stimulating mechanism. Here, the non-small cell lung cancer (NSCLC) PC9 cell line was used to systematically profile, by mass spectrometry, changes in the proteome, kinome, and phosphoproteome during early treatment with the EGFR inhibitor afatinib. Regardless of the response, initial drug-sensitive cells rapidly adapt to targeted therapy, and within days, cells regained the capacity to proliferate, despite persisting target inhibition. These data reveal a rapid reactivation of mTOR and MAPK signaling pathways after initial inhibition and an increase in abundance and activity of cytoskeleton and calcium signaling-related proteins. Pharmacologic inhibition of reactivated pathways resulted in increased afatinib efficacy. However more strikingly, cells that were restricted from accessing extracellular calcium were extremely sensitive to afatinib treatment. These findings were validated using three additional inhibitors tested in four different NSCLC cell lines, and the data clearly indicated a role for Ca signaling during the development of adaptive resistance. From a therapeutic point of view, the increased inhibitor efficacy could limit or even prevent further resistance development. Combined targeting of calcium signaling and RTKs may limit drug resistance and improve treatment efficacy. .

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