Due to the dynamic nature and low stoichiometry of protein phosphorylation, enrichment of phosphorylated peptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Several phosphopeptide isolation strategies have been presented in the literature, including immobilized metal ion affinity chromatography. However, that technique suffers from poor selectivity and reproducibility. Recently, titanium dioxide-based columns have been successfully employed for phosphopeptide enrichment by several research groups. Here, we present, to our knowledge, the first demonstration of the utility of zirconium dioxide microtips for phosphopeptide isolation prior to mass spectrometric analysis. These microtips display similar overall performance as TiO2 microtips. However, more selective isolation of singly phosphorylated peptides was observed with ZrO2 compared to TiO2 whereas TiO2 preferentially enriched multiply phosphorylated peptides. Thus, these two chromatographic materials possess complementary properties. For alpha- and beta-casein, Glu-C digestion provided no evident advantage compared to trypsin digestion when combined with TiO2 or ZrO2 phosphopeptide enrichment.

In many macroorganisms, the ultimate source of potent biologically active natural products has remained elusive due to an inability to identify and culture the producing symbiotic microorganisms. As a model system for developing a meta-omic approach to identify and characterize natural product pathways from invertebrate-derived microbial consortia we chose to investigate the ET-743 (Yondelis®) biosynthetic pathway. This molecule is an approved anti-cancer agent obtained in low abundance (10−4–10−5% w/w) from the tunicate Ecteinascidia turbinata, and is generated in suitable quantities for clinical use by a lengthy semi-synthetic process. Based on structural similarities to three bacterial secondary metabolites, we hypothesized that ET-743 is the product of a marine bacterial symbiont. Using metagenomic sequencing of total DNA from the tunicate/microbial consortium we targeted and assembled a 35 kb contig containing 25 genes that comprise the core of the NRPS biosynthetic pathway for this valuable anti-cancer agent. Rigorous sequence analysis based on codon usage of two large unlinked contigs suggests that Candidatus Endoecteinascidia frumentensis produces the ET-743 metabolite. Subsequent metaproteomic analysis confirmed expression of three key biosynthetic proteins. Moreover, the predicted activity of an enzyme for assembly of the tetrahydroisoquinoline core of ET-743 was verified in vitro. This work provides a foundation for direct production of the drug and new analogs through metabolic engineering. We expect that the interdisciplinary approach described is applicable to diverse host-symbiont systems that generate valuable natural products for drug discovery and development.

The mammalian homologs of the C. elegans partitioning-defective (Par) proteins have been demonstrated to be necessary for establishment of cell polarity. In mammalian epithelia, the Par3/Par6/aPKC polarity complex is localized to the tight junction and regulates its formation and positioning with respect to basolateral and apical membrane domains. Here we demonstrate a previously undescribed phosphorylation-dependent interaction between a mammalian homolog of the C. elegans polarity protein Par5, 14-3-3, and the tight junction-associated protein Par3. We identify phosphorylated serine 144 as a site of 14-3-3 binding. Expression of a Par3 mutant that contains serine 144 mutated to alanine (S144A) results in defects in epithelial cell polarity. In addition, overexpression of 14-3-3zeta results in a severe disruption of polarity, whereas overexpression of a 14-3-3 mutant that is defective in binding to phosphoproteins has no effect on cell polarity. Together, these data suggest a novel, phosphorylation-dependent mechanism that regulates the function of the Par3/Par6/aPKC polarity complex through 14-3-3 binding.

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Gas-phase ion-electron reactions, including electron capture dissociation (ECD) and electron detachment dissociation (EDD), are advantageous for characterization of protein posttranslational modifications (PTMs), because labile modifications are not lost during the fragmentation process. However, at least two positive charges and relatively abundant precursor ions are required for ECD due to charge reduction and lower fragmentation efficiency compared to conventional gas-phase fragmentation techniques. Both these criteria are difficult to fulfill for phosphopeptides due to their acidic character. The negative ion mode operation of EDD is more compatible with phosphopeptide ionization, but EDD suffers from a fragmentation efficiency even lower than that of ECD. Recently, metal oxides such as ZrO 2 and TiO 2 have been shown to provide selective enrichment of phosphopeptides from proteolytic digests. Here, we utilize this enrichment strategy to improve ECD and EDD of phosphopeptides. This approach allowed determination of the locations of phosphorylation sites in highly acidic, multiply phosphorylated peptides from complex peptide mixtures by ECD. For singly phosphorylated peptides, EDD provided complementary sequence information compared to ECD.