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Significance Protein phosphorylation is a major regulatory mechanism for many cellular functions, but no phosphoprotein in biofluids has been developed for disease diagnosis because of the presence of active phosphatases. This study presents a general strategy to isolate and identify phosphoproteins in extracellular vesicles (EVs) from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides in EVs from small volumes of plasma samples and more than 100 phosphoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer as compared with healthy controls. This study demonstrates that the development of phosphoproteins in plasma EVs as disease biomarkers is highly feasible and may transform cancer screening and monitoring.

Exotoxins, including the hemolysins known as the alpha (α) and beta (β) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding α toxin was decreased in a Δstp1 mutant strain and increased in a Δstk1 strain. Microarray analysis of a Δstk1 mutant revealed increased transcription of additional exotoxins. A Δstp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Δstk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Δstk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence.

The ability to obtain in-depth understanding of signaling networks in cells is a key objective of systems biology research. Such ability depends largely on unbiased and reproducible analysis of phosphoproteomes. We present here a novel proteomics tool, polymer-based metal ion affinity capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate comprehensive phosphoproteome analyses. This approach uses polyamidoamine dendrimers multifunctionalized with titanium ions and aldehyde groups to allow the chelation and subsequent isolation of phosphopeptides in a homogeneous environment. Compared with current strategies based on solid phase micro-and nanoparticles, PolyMAC demonstrated outstanding reproducibility, exceptional selectivity, fast chelation times, and high phosphopeptide recovery from complex mixtures. Using the PolyMAC method combined with antibody enrichment, we identified 794 unique sites of tyrosine phosphorylation in malignant breast cancer cells, 514 of which are dependent on the expression of Syk, a protein-tyrosine kinase with unusual properties of a tumor suppressor. The superior sensitivity of PolyMAC allowed us to identify novel components in a variety of major signaling networks, including cell migration and apoptosis. PolyMAC offers a powerful and widely applicable tool for phosphoproteomics and molecular signaling. Molecular & Cellular Proteomics 9:2162-2172, 2010.Reversible phosphorylation of proteins is a major mechanism for the regulation of multiple cellular processes (1, 2). Mass spectrometry-based phosphoproteomics provides a method for the global analysis of protein phosphorylation and molecular signaling in cells (3,4). Despite the great progress that has been made over the past few years, the isolation of phosphopeptides and their analysis by mass spectrometry are still a considerable challenge because of the typically low stoichiometry of protein phosphorylation and the resulting low abundance of phosphopeptides. An early step in any phosphoproteome analysis is the isolation of phosphopeptides, preferably with high efficiency, selectivity, sensitivity, and reproducibility. Currently, there are three major strategies for the isolation of phosphopeptides: antibody-based affinity capture, chemical derivatization of phosphoamino acids, and metal ion-based affinity capture. Antibody-based methods are used mainly for the selective isolation of phosphotyrosinecontaining proteins or peptides (5-8). Chemical derivatization methods begin with the ␤-elimination of phosphates from phosphoserine and phosphothreonine (9) or the formation of phosphoramidates by reactions with amines (10) to selectively immobilize phosphopeptides. Metal ion-based affinity capture techniques use immobilized metal affinity chromatography (IMAC) with Fe(III) (11,12) or Ga(III) (13) and, for the past a few years, more successful metal oxide approaches (i.e. TiO 2 (14, 15) and ZrO 2 (16, 17)) for the selective binding of phosphorylated peptides. Almost all of the current isolation methods are b...