In this work, for the first time, Ti(4+)-Fe3O4@polydopamine microspheres were designed and synthesized for efficient and selective enrichment of phosphopeptides in biological samples.

To discover trace phosphorylated proteins or peptides with great biological significance for in-depth phosphoproteome analysis, it is urgent to develop a novel technique for highly selective and effective enrichment of phosphopeptides. In this work, an IMAC (immobilized metal ion affinity chromatography) material with polydopamine coated on the surface of graphene and functionalized with titanium ions (denoted as Ti(4+)-G@PD) was initially designed and synthesized. The newly prepared Ti(4+)-G@PD with enhanced hydrophilicity and biological compatibility was characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and infrared (IR), and its performance for selective and effective enrichment of phosphopeptide was evaluated with both standard peptide mixtures and human serum.

Human toxic responses are very often related to metabolism. Liver metabolism is traditionally studied, but other organs also convert chemicals and drugs to reactive metabolites leading to toxicity. When DNA damage is found, the effects are termed genotoxic. Here we describe a comprehensive new approach to evaluate chemical genotoxicity pathways from metabolites formed in-situ by a broad spectrum of liver, lung, kidney and intestinal enzymes. DNA damage rates are measured with a microfluidic array featuring a 64-nanowell chip to facilitate fabrication of films of DNA, electrochemiluminescent (ECL) detection polymer [Ru(bpy)2(PVP)10]2+ {(PVP = poly(4-vinylpyridine)} and metabolic enzymes. First, multiple enzyme reactions are run on test compounds using the array, then ECL light related to the resulting DNA damage is measured. A companion method next facilitates reaction of target compounds with DNA/enzyme-coated magnetic beads in 96 well plates, after which DNA is hydrolyzed and nucleobase-metabolite adducts are detected by LC-MS/MS. The same organ enzymes are used as in the arrays. Outcomes revealed nucleobase adducts from DNA damage, enzymes responsible for reactive metabolites (e.g. cyt P450s), influence of bioconjugation, relative dynamics of enzymes suites from different organs, and pathways of possible genotoxic chemistry. Correlations between DNA damage rates from the cell-free array and organ-specific cell-based DNA damage were found. Results illustrate the power of the combined DNA/enzyme microarray/LC-MS/MS approach to efficiently explore a broad spectrum of organ-specific metabolic genotoxic pathways for drugs and environmental chemicals.

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