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We have tested the effect of m-nitrobenzyl alcohol (m-NBA) as a method to increase the average charge state of protonated gas-phase molecular ions generated by ESI from tryptic peptides and phosphopeptides. Various concentrations of m-NBA were added to the mobile phases of a liquid chromatography system coupled to an ESI tandem mass spectrometer. Addition of just 0.1% m-NBA changed the average charge state for the identified tryptic BSA peptides from 2.2+ to 2.6+. As a result, the predominant charge states for BSA peptides were changed from 2+ to [greater than or equal to] 3+. To evaluate the benefits of peptide charge enhancement, the ETD fragmentation efficiency and Mascot peptide score were compared for BSA peptides in charge states 2+ and 3+. In all cases but one, triply charged peptides fragmented more efficiently than the analogues 2+ peptide ions. On average, triply charged peptides received a 68% higher Mascot score (24 units) than doubly charged peptides, m-NBA also increased the average charge state of phosphopeptides by up to 0.5 charge unit. The ease of implementation and the analytical benefits of charge enhancement of tryptic peptides by addition of m-NBA to the LC solvents suggest the general application of this reagent in proteomic studies that employ ETD-MS/MS and related techniques.

We tested for oxidative coupling of phenolic materials, including organic contaminants and tyrosine, in gut fluid from marine deposit-feeding invertebrates. A phenolic metabolite (pyrenol) of a polycyclic aromatic hydrocarbon (PAH), which can arise during accumulation by deposit-feeding invertebrates, was found to participate in oxidative coupling reactions to organic matter in gut fluid. Gut fluid from four species of marine deposit-feeding invertebrates (three polychaetes and one holothuroid) catalyzed oxidative coupling of pyrenol (1-hydroxypyrene) in rough proportion to their enzyme activity and dissolved organic matter levels. Nereis virens gut fluid also catalyzed coupling of tyrosine monomers to form dityrosine, a common marker of oxidative damage in proteins. An antioxidant enzyme activity, similar to that of heme peroxidase and capable of oxidative coupling, was tentatively identified in gut fluid from N. virens by protein precipitation, dye decolorization assays, and enzyme inhibitor studies. Unaltered N. virens gut fluid had high total oxyradical scavenging capacity, indicating the presence of fast-acting antioxidants. Oxidative coupling of PAHs will reduce subsequent bioavailability, toxicity, and transport of these compounds in marine environments. Furthermore, oxidative coupling of PAHs represents a hitherto overlooked sink for organic contaminants in marine sediments and suggests a biological mechanism for formation of aquatic humic material.