Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox

High molecular weight homologues of gp91phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for “dual oxidase” because they have both a peroxidase homology domain and a gp91phox domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals. To investigate function, RNA interference (RNAi) was carried out in C. elegans. RNAi animals showed complex phenotypes similar to those described previously in mutations in collagen biosynthesis that are known to affect the cuticle, an extracellular matrix. Electron micrographs showed gross abnormalities in the cuticle of RNAi animals. In cuticle, collagen and other proteins are cross-linked via di- and trityrosine linkages, and these linkages were absent in RNAi animals. The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed cross-linking of free tyrosine ethyl ester. Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix

An Introduction to Sphingolipid Metabolism and Analysis by New Technologies

Sphingolipids (SP) are a complex class of molecules found in essentially all eukaryotes and some prokaryotes and viruses where they influence membrane structure, intracellular signaling, and interactions with the extracellular environment. Because of the combinatorial nature of their biosynthesis, there are thousands of SP subspecies varying in the lipid backbones and complex phospho- and glycoheadgroups. Therefore, comprehensive or “sphingolipidomic” analyses (structure-specific, quantitative analyses of all SP, or at least all members of a critical subset) are needed to know which and how much of these subspecies are present in a system as a step toward understanding their functions. Mass spectrometry and related novel techniques are able to quantify a small fraction, but nonetheless a substantial number, of SP and are beginning to provide information about their localization. This review summarizes the basic metabolism of SP and state-of-art mass spectrometric techniques that are producing insights into SP structure, metabolism, functions, and some of the dysfunctions of relevance to neuromedicine

On the use of scans at a constant ratio of B/E for studying decompositions of peptide metal(II)-ion complexes formed by electrospray ionization

The use of sector mass spectrometers to study metastable ion decompositions of peptide metal-ion complexes formed by electrospray ionization is discussed. Products that are formed by charge-separation reactions are characterized by large kinetic energy release distributions. This causes scans at a constant B/E to give incorrect product ion abundances and possibly incorrect mass assignments. Two instrumental methods exist that can be used either to detect the ions or to estimate relative ion abundances: a floated collision cell or mass-analyzed ion kinetic energy spectrometry (MIKES) scans. The floated collision cell, by virtue of an altered B/E scan law, however, discriminates against important metastable ion reactions that occur outside the cell. MIKES scans provide a clearer estimate of product ions that arise by metastable ion charge-separation reactions. Problems with pseudotandem (first field-free region) experiments are also discussed

Serum biomarker profiling by solid‐phase extraction with particle‐embedded micro tips and matrix‐assisted laser desorption/ionization mass spectrometry

One of the main challenges in high-throughput serum profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the development of proteome fractionation approaches that allow the acquisition of reproducible profiles with a maximum number of spectral features and minimum interferences from biological matrices. This study evaluates a new class of solid-phase extraction (SPE) pipette tips embedded with different chromatographic media for fractionation of model protein digests and serum samples. The materials embedded include strong anion exchange (SAX), weak cation exchange (WCX), C18, C8, C4, immobilized metal affinity chromatography (IMAC) and zirconium dioxide particles. Simple and rapid serum proteome profiling protocols based on these SPE micro tips are described and tested using a variety of MALDI matrices. We show that different types of particle-embedded SPE micro tips provide complementary information in terms of the spectral features detected for β-casein digests and control human serum samples. The effect of different sample pretreatments, such as serum dilution and ultrafiltration using molecular weight cut-off membranes, and the reproducibility observed for replicate experiments, are also evaluated. The results demonstrate the usefulness of these simple SPE tips combined with offline MALDI-TOF MS for obtaining information-rich serum profiles, resulting in a robust, versatile and reproducible open-source platform for serum biomarker discovery