Ommochrome Wing Pigments in the Monarch Butterfly Danaus plexippus (Lepidoptera: Nymphalidae).

Monarch butterflies (Danaus plexippus) use bright orange coloration to warn off predators as well as for sexual selection. Surprisingly the underlying pigment compounds have not been previously characterized. We used LCMS and fragmentation MS (including MSMS and MSn) of extracted pigments from nonmigratory summer-generation female monarch forewings to identify and provide relative quantitation of various orange pigments from D.

Novel "omics" approach for study of low-abundance, low-molecular-weight components of a complex biological tissue: regional differences between chorionic and basal plates of the human placenta.

Tissue proteomics has relied heavily on two-dimensional gel electrophoresis, for protein separation and quantification, then single protein isolation, trypsin digestion, and mass spectrometric protein identification. Such methods are predominantly used for study of high-abundance, full-length proteins. Tissue peptidomics has recently been developed but is still used to study the most highly abundant species, often resulting in observation and identification of dozens of peptides only.

Tissue proteomics of the low-molecular weight proteome using an integrated cLC-ESI-QTOFMS approach.

Analysis of the protein/peptide composition of tissue has provided meaningful insights into tissue biology and even disease mechanisms. However, little has been published regarding top down methods to investigate lower molecular weight (MW) (500-5000 Da) species in tissue. Here, we evaluate a tissue proteomics approach involving tissue homogenization followed by depletion of large proteins and then cLC-MS (where c stands for capillary) analysis to interrogate the low MW/low abundance tissue proteome.

An integrated serum proteomic approach capable of monitoring the low molecular weight proteome with sequencing of intermediate to large peptides.

The low-abundance, low molecular weight serum proteome has high potential for the discovery of new biomarkers using mass spectrometry (MS). Because the serum proteome is large and complex, defining relative quantitative differences for a molecular species between comparison groups requires an approach with robust separation capability, high sensitivity, as well as high mass resolution. Capillary liquid chromatography (cLC)/MS provides both the necessary separation technique and the sensitivity to observe many low-abundance peptides.

Systematic internal standard selection for capillary liquid chromatography-mass spectrometry time normalization to facilitate serum proteomics.

Because blood interacts with almost all tissues of the body, it is likely that changes in the overall health of an organism will be reflected in the quantities of specific serum peptides and proteins, making them biomarkers. Due to the complexity of serum, pre-analytical sample simplification and separation are needed prior to mass spectrometric analysis. Use of a reverse-phase capillary column coupled to a mass spectrometer allows for separation and analysis of serum as part of efforts to discover biomarkers.

Influence of diet on the proteome of Drosophila melanogaster as assessed by two-dimensional gel electrophoresis and capillary liquid chromatography-mass spectrometry: the hamburger effect revisited.

Proteomic biomarker discovery has been called into question. Diamandis hypothesized that seemingly trivial factors, such as eating a hamburger, may cause sufficient proteomic change as to confound proteomic differences. This has been termed the hamburger effect.

Screening phosphatidylcholine biomarkers in mouse liver extracts from a hypercholesterolemia study using ESI-MS and chemometrics.

When fed a high-fat, high-cholesterol diet (HFD), homozygous LDL receptor knockout mice exhibit extremely high levels of plasma cholesterol that are expected to influence liver metabolism. One step in the investigation of potential hepatic alterations was the analysis of organic extracts of livers from these and control mice by electrospray mass spectrometry (ESI-MS). Chemometrics (bioinformatics) analysis shows that the sample spectra cluster into two groups: one from mice with plasma cholesterol levels in excess of 900 mg dL(-1) and one from animals with cholesterol levels of 60-250 mg dL(-1).

Proteomic and phototoxic characterization of melanolipofuscin: correlation to disease and model for its origin.

Purpose: Melanolipofuscin (MLF) is a complex granule, exhibiting properties of both melanosomes and lipofuscin (LF) granules, which accumulates in retinal pigment epithelial (RPE) cells and may contribute to the etiology of age-related macular degeneration (AMD). MLF accumulation has been reported by Feeney-Burns to more closely reflect the onset of AMD than the accumulation of lipofuscin. In an effort to assess the possible contribution MLF may have to the onset of AMD, we analyzed the phototoxicity and protein composition of MLF and compared those results to that of LF.

Coupled affinity-hydrophobic monolithic column for on-line removal of immunoglobulin G, preconcentration of low abundance proteins and separation by capillary zone electrophoresis.

A butyl methacrylate-co-ethylene dimethacrylate (BuMA-co-EDMA) monolith was synthesized by UV initiated polymerization at the inlet end of a 75 microm I.D. fused silica capillary that had been previously coated with a protein compatible polymer, poly(vinyl)alcohol.

Efficient polymer monolith for strong cation-exchange capillary liquid chromatography of peptides.

A stable poly[2-acrylamido-2-methyl-1-propanesulfonic acid-co-poly(ethylene glycol) diacrylate] monolith was synthesized inside a 75-microm-i.d. capillary by photoinitiated copolymerization with water, methanol, and ethyl ether as porogens.

Mechanism of assembly of G protein betagamma subunits by protein kinase CK2-phosphorylated phosducin-like protein and the cytosolic chaperonin complex.

Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein betagamma subunit complex (Gbetagamma) that has been recently shown to catalyze the formation of the Gbetagamma dimer from its nascent polypeptides. Phosphorylation of PhLP at one or more of three consecutive serines (Ser-18, Ser-19, and Ser-20) is necessary for Gbetagamma dimer formation and is believed to be mediated by the protein kinase CK2. Moreover, several lines of evidence suggest that the cytosolic chaperonin complex (CCT) may work in concert with PhLP in the Gbetagamma-assembly process.

Examining the proteins of functional retinal lipofuscin using proteomic analysis as a guide for understanding its origin.

Purpose: To elucidate the origins of biologically active retinal lipofuscin (RLF) by examining its protein composition.

Methods: Total protein and total lipid were extracted and quantified. Proteins in this lipoprotein granule were identified by limited-scale proteomic analysis using both two-dimensional (2D) gel electrophoresis and SDS-PAGE coupled with MALDI-QqToF MSMS and automated LCMSMS, respectively.

Design and evaluation of a coupled monolithic pre-concentrator-capillary zone electrophoresis system for the extraction of immunoglobulin G from human serum.

The analysis of proteins in biological fluids by capillary electrophoresis (CE) is of interest in clinical chemistry. However, due to low analyte concentrations and poor concentration limits of detection (CLOD), protein analysis by this technique is frequently challenging. Coupling preconcentration techniques with CE greatly improves the CLOD.

Identification of phosphorylation sites on phosducin-like protein by QTOF mass spectrometry.

Post-translational modifications are used by cells to control the functions of proteins. Phosducin-like protein (PhLP) is a regulator of G-protein signaling that is post-translationally modified via phosphorylation. Phosphorylation of PhLP initiates its degradation by the 26S proteasome in serum-stimulated cells.

Initial analysis of the phosphoproteome of Chinese hamster ovary cells using electrophoresis.

Protein phosphorylation is a common post-translational modification of enormous biological importance. Analysis of phosphorylation at the global level should shed light on the use of this modification to regulate metabolism, signal transduction, and other processes. We have begun a proteomic analysis of phosphorylation using two-dimensional gel electrophoresis.

Analysis of low-abundance, low-molecular-weight serum proteins using mass spectrometry.

To detect diseases early in the general population, new diagnostic approaches are needed that have adequate sensitivity and specificity. Recent studies have used mass spectrometry to identify a serum proteomic pattern for breast and ovarian cancer. Serum contains 60-80 mg/mL protein, but 57-71% of this is serum albumin, and 8-26% are gamma-globulins.

Site-specific phosphorylation of phosducin in intact retina. Dynamics of phosphorylation and effects on G protein beta gamma dimer binding.

Phosducin (Pdc) is a G protein beta gamma dimer (G beta gamma) binding protein, highly expressed in retinal photoreceptor and pineal cells, yet whose physiological role remains elusive. Light controls the phosphorylation of Pdc in a cAMP and Ca(2+)-dependent manner, and phosphorylation in turn regulates the binding of Pdc to G(t)beta gamma or 14-3-3 proteins in vitro. To directly examine the phosphorylation of Pdc in intact retina, we prepared antibodies specific to the three principal phosphorylation sites (Ser-54, Ser-73, and Ser-106) and measured the kinetics of phosphorylation/dephosphorylation during light/dark adaptation and the subsequent effects on G(t)beta gamma binding.

Identification and characterization of a Pi isoform of glutathione S-transferase (GSTP1) as a zeaxanthin-binding protein in the macula of the human eye.

Uptake, metabolism, and stabilization of xanthophyll carotenoids in the retina are thought to be mediated by specific xanthophyll-binding proteins (XBPs). A membrane-associated XBP was purified from human macula using ion-exchange chromatography followed by gel-exclusion chromatography. Two-dimensional gel electrophoresis showed a prominent spot of 23 kDa and an isoelectric point of 5.

Regulation of angiotensin II-induced G protein signaling by phosducin-like protein.

Phosducin-like protein (PhLP) is a broadly expressed member of the phosducin (Pd) family of G protein betagamma subunit (Gbetagamma)-binding proteins. Though PhLP has been shown to bind Gbetagamma in vitro, little is known about its physiological function. In the present study, the effect of PhLP on angiotensin II (Ang II) signaling was measured in Chinese hamster ovary cells expressing the type 1 Ang II receptor and various amounts of PhLP.

Regulatory interaction of phosducin-like protein with the cytosolic chaperonin complex.

Phosducin and phosducin-like protein (PhLP) bind G protein betagamma subunits and regulate their activity. This report describes a previously uncharacterized binding partner unique to PhLP that was discovered by coimmunoprecipitation coupled with mass spectrometric identification. Chaperonin containing tailless complex polypeptide 1 (CCT), a cytosolic chaperone responsible for the folding of many cellular proteins, binds PhLP with a stoichiometry of one PhLP per CCT complex.