The 2012/2013 ABRF Proteomic Research Group Study: Assessing Longitudinal Intralaboratory Variability in Routine Peptide Liquid Chromatography Tandem Mass Spectrometry Analyses.

Questions concerning longitudinal data quality and reproducibility of proteomic laboratories spurred the Protein Research Group of the Association of Biomolecular Resource Facilities (ABRF-PRG) to design a study to systematically assess the reproducibility of proteomic laboratories over an extended period of time. Developed as an open study, initially 64 participants were recruited from the broader mass spectrometry community to analyze provided aliquots of a six bovine protein tryptic digest mixture every month for a period of nine months. Data were uploaded to a central repository, and the operators answered an accompanying survey.

An enhanced throughput method for quantification of sulfur mustard adducts to human serum albumin via isotope dilution tandem mass spectrometry.

Here, we report an enhanced throughput method for the diagnosis of human exposure to sulfur mustard. A hydroxyethylthioethyl (HETE) ester-adducted tripeptide, produced by pronase digestion of human serum albumin, was selected as the quantitative exposure biomarker. Cibacron Blue enrichment was developed from an established cartridge method into a 96-well plate format, increasing throughput and ruggedness.

Selective inhibition of cell proliferation by lycopene in MCF-7 breast cancer cells in vitro: a proteomic analysis.

Lycopene, a red pigmented carotenoid present in many fruits and vegetables such as tomatoes, has been associated with the reduced risk of breast cancer. This study sought to identify proteins modulated by lycopene during cell proliferation of the breast cancer cell line MCF-7 to gain an understanding into its mechanism of action. MCF-7 breast cancer cells and MCF-10 normal breast cells were treated with 0, 2, 4, 6, 8, and 10 μM of lycopene for 72 h.

The ABRF Proteomics Research Group studies: educational exercises for qualitative and quantitative proteomic analyses.

Resource (core) facilities have played an ever-increasing role in furnishing the scientific community with specialized instrumentation and expertise for proteomics experiments in a cost-effective manner. The Proteomics Research Group (PRG) of the Association of Biomolecular Resource Facilities (ABRF) has sponsored a number of research studies designed to enable participants to try new techniques and assess their capabilities relative to other laboratories analyzing the same samples. Presented here are results from three PRG studies representing different samples that are typically analyzed in a core facility, ranging from simple protein identification to targeted analyses, and include intentional challenges to reflect realistic studies.

Population proteomics: quantitative variation within and among populations in cardiac protein expression.

Population analysis of gene expression is typically achieved by quantifying levels of mRNA; however, gene expression is also a function of protein translation and turnover. Therefore, a complete understanding of population variation in gene expression requires quantitative knowledge of protein expression within and among natural populations. We used two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) to quantitatively compare expression of heart ventricle proteins among 18 individuals in three populations of the teleost fish Fundulus.

Leptin modulated changes in adipose tissue protein expression in ob/ob mice.

Comparative proteomic analyses were performed in adipose tissue of leptin-deficient ob/ob mice treated with leptin or control buffer in order to identify the protein expression changes as the potential targets of leptin. Mice were treated with either phosphate-buffered saline (control) or 10 µg/day leptin for 14 days via subcutaneous osmotic minipumps. Total protein from white adipose tissue was extracted and labeled with different fluorescent cyanine dyes for analysis by two-dimensional difference gel electrophoresis (DIGE).

Proteomic analysis of Alzheimer's disease cerebrospinal fluid from neuropathologically diagnosed subjects.

A crucial need exists for reliable Alzheimer's disease (AD) diagnostic and prognostic tests. Given its intimate communication with the brain, the cerebrospinal fluid (CSF) has been surveyed intensively for reliable AD biomarkers. The heterogeneity of AD pathology and the unavoidable difficulties associated with the clinical diagnosis and differentiation of this dementia from other pathologies have confounded biomarker studies in antemortem CSF samples.

Proteomic identification of Bacillus thuringiensis subsp. israelensis toxin Cry4Ba binding proteins in midgut membranes from Aedes (Stegomyia) aegypti Linnaeus (Diptera, Culicidae) larvae.

Novel Bacillus thuringiensis subsp. israelensis (Bti) Cry4Ba toxin-binding proteins have been identified in gut brush border membranes of the Aedes (Stegomyia) aegypti mosquito larvae by combining 2-dimensional gel electrophoresis (2DE) and ligand blotting followed by protein identification using mass spectrometry and database searching. Three alkaline phosphatase isoforms and aminopeptidase were identified.

A 106-kDa aminopeptidase is a putative receptor for Bacillus thuringiensis Cry11Ba toxin in the mosquito Anopheles gambiae.

Bacillus thuringiensis (Bt) insecticidal toxins bind to receptors on midgut epithelial cells of susceptible insects, and binding triggers biochemical events that lead to insect mortality. Recently, a 100-kDa aminopeptidase N (APN) was isolated from brush border membrane vesicles (BBMV) of Anopheles quadrimaculatus and shown to bind Cry11Ba toxin with surface plasmon resonance (SPR) detection [Abdullah et al. (2006) BMC Biochem.

Genomic differences between Campylobacter jejuni isolates identify surface membrane and flagellar function gene products potentially important for colonizing the chicken intestine.

Campylobacter spp. are one of the leading bacterial etiologic agents of acute human gastroenteritis among industrialized countries. Poultry are implicated as a major source of the organism for human illness; however, the factors involved with colonization of poultry gastrointestinal systems remain unclear.

Identification of major sporulation proteins of Myxococcus xanthus using a proteomic approach.

Myxococcus xanthus is a soil-dwelling, gram-negative bacterium that during nutrient deprivation is capable of undergoing morphogenesis from a vegetative rod to a spherical, stress-resistant spore inside a domed-shaped, multicellular fruiting body. To identify proteins required for building stress-resistant M. xanthus spores, we compared the proteome of liquid-grown vegetative cells with the proteome of mature fruiting body spores.

Effect of staining reagent on peptide mass fingerprinting from in-gel trypsin digestions: a comparison of SyproRuby and DeepPurple.

As the new fluorescent stains such as SyproRuby and DeepPurple are getting widespread recognition for proteome analyses by the traditional 2-D gel method, it becomes important to test the feasibility of these stains with respect to staining reproducibility, protein quantitation, and compatibility of the stain with downstream MS. The binding of epicocconone, active ingredient of DeepPurple, to one of the primary cleavage sites of trypsin (lysine residue) raises the possibility of incomplete cleavage and interference with PMF. However, the current study tests and concludes that the DeepPurple stain can result in increased peptide recovery compared to SyproRuby stain and can improve MS-based identification of lower intensity proteins spots.

New frontiers in proteomics research: a perspective.

Substantial advances have been made in the fundamental understanding of human biology, ranging from DNA structure to identification of diseases associated with genetic abnormalities. Genome sequence information is becoming available in unprecedented amounts. The absence of a direct functional correlation between gene transcripts and their corresponding proteins, however, represents a significant roadblock for improving the efficiency of biological discoveries.

Rapid and improved method for windowing eggs accessing the stage X chicken embryo.

The chick stage X blastoderm is routinely accessed through a small window in a freshly laid egg. However, windowing severely compromises embryo survival with hatch rates as low as a few percent. We previously reported a simple modification to the standard method that reduced introduction of air into the sealed egg and improved the hatchability to 32%.