Project description:Hydroxyl radical protein footprinting (HRPF) is a powerful technique for probing changes in protein topography, based on quantifying the amount of oxidation of different regions of a protein. While quantification of HRPF oxidation at the peptide level is relatively common and straightforward, quantification at the residue level is challenging because of the influence of oxidation on MS/MS fragmentation and the large number of complex and only partially chromatographically resolved isomeric peptide oxidation products. HRPF quantification of isomeric peptide oxidation products (where the peptide sequence is the same but isomeric oxidation products are formed at different sites) at the residue level by electron transfer dissociation tandem mass spectrometry (ETD MS/MS) has been demonstrated in both model peptides and HRPF products, but the method is hampered by the partial separation of oxidation isomers by reversed phase chromatography. This requires custom MS/MS methods to equally sample all isomeric oxidation products across their elution window, greatly increasing method development time and reducing the oxidation products quantified in a single LC-MS/MS run. Here, we present a zwitterionic hydrophilic interaction capillary chromatography (ZIC-HILIC) method to ideally coelute all isomeric peptide oxidation products while separating different peptides. This allows us to relatively quantify peptide oxidation isomers using an ETD MS/MS spectrum acquired at any point across the single peptide oxidation isomer peak, greatly simplifying data acquisition and data analysis.
Project description:The number and type of synthetic chemicals that are being produced worldwide continues to increase significantly. While these industrial chemicals provide numerous benefits, there is no doubt that some have potential to damage the environment and health. Toxicity must be evaluated and use must be carefully controlled and monitored in order to minimize potential damage. DNA microarray technology has become an important new technique in toxicology. We are using the yeast Saccharomyces cerevisiae as a model organism for toxicological study because it is a simple, fast-growing eukaryote that has been thoroughly characterized. In order to evaluate toxicity by newly synthesized or mixture chemicals, toxicity-induced gene expression alteration profiles by known chemicals should be collected. Nitrophenols belong to the family of nitro compounds. There are three isomers, depending upon position of the functional groups at the aromatic ring: o- (CAS; 88-75-5), m- (CAS; 554-84-7) and p- (CAS; 100-02-7). It was reported that 4-nitrophenol is reported to be more toxic than 2- in animal test. In our yeast result, IC50 of o-, m- and p-nitrophenol was 3 mM, 5 mM, 1.5 mM, respectively, indicating highest toxic isomer was p-. p-Nitrophenol is reported to cause methemoglobinemia, and all isomers are suspected to be cardiovascular or blood toxicant, neurotoxicant. Keywords: stress response
Project description:Dechlorination of three tetrachlorobenzene isomers by enrichment cultures originating from a contaminated harbor follows thermodynamically favorable reactions
Project description:Reversible protein phosphorylation is an essential regulatory component of virtually every cellular process and is frequently dysregulated in cancer. However, significant analytical barriers persist that hamper the routine application of phosphoproteomics in translational settings. Here, we present a straightforward and reproducible approach for the broadscale analysis of protein phosphorylation that relies on a single phosphopeptide enrichment step using titanium dioxide microspheres from whole cell lysate digests and compared it to the well-established SCX-TiO(2) workflow for phosphopeptide purification on a proteome-wide scale. We demonstrate the scaleabilty of our approach from 200 μg to 5 mg of total NCI-H23 non-small cell lung adenocarcinoma cell lysate digest and determine its quantitative reproducibility by label-free analysis of phosphopeptide peak areas from replicate purifications (median CV: 20% RSD). Finally, we combine this approach with immunoaffinity phosphotyrosine enrichment, enabling the identification of 3168 unique nonredundant phosphotyrosine peptides in two LC-MS/MS runs from 8 mg of HeLa peptides, each with 80% phosphotyrosine selectivity, at a peptide FDR of 0.2%. Taken together, we establish and validate a robust approach for proteome-wide phosphorylation analysis in a variety of scenarios that is easy to implement in biomedical research and translational settings.
Project description:Multiple types of natural collagens specifically assemble and co-exist in the extracellular matrix. Although noncollagenous trimerization domains facilitate the folding of triple-helical regions, it is intriguing to ask whether collagen sequences are also capable of controlling heterospecific association. In this study, we designed a model system mimicking simultaneous specific assembly of two collagen heterotrimers using a genetically inspired operation, circular permutation. Previously, surface charge-pair interactions were optimized on three collagen peptides to promote the formation of an abc-type heterotrimer. Circular permutation of these sequences retained networks of stabilizing interactions, preserving both triple-helical structure and heterospecificity of assembly. Combining original peptides A, B, and C and permuted peptides D, E, and F resulted primarily in formation of A:B:C and D:E:F, a heterospecificity of 2 of 56 possible stoichiometries. This degree of specificity in collagen molecular recognition is unprecedented in natural or synthetic collagens. Analysis of natural collagen sequences indicates low similarity between the neighboring exons. Combining the synthetic collagen model and bioinformatic analysis provides insight on how fibrillar collagens might have arisen from the duplication of smaller domains.
Project description:Objective: To quantify changes in adipogenic gene expression in the presence of ritonavir (RTV) or tenofovir (TDF), and determine whether conjugated linoleic acid (CLA) isomers (cis9,trans11 or trans10,cis12) can mitigate detrimental effects of antiretoviral drugs. Methods: Affymetrix Mouse Genome 430 2.0 microarray was used to investigate gene expression in 3T3-L1 adipocytes treated with (1) RTV, TDF or ethanol control, or (2) ritonavir +c9,t11-CLA, ritonavir+t10,c12-CLA or ritonavir+DMSO control. RT-PCR validation of Pparg, Adipoq and Retn was carried out. ELISA and DNA binding ELISA were used to investigate secreted proteins and Pparg binding to its gene response element. Oil Red O staining was used to investigate triglyceride accumulation. Results: No effect was observed for TDF. Expression of 389 genes was altered more than 5-fold in the presence of RTV. Down-regulated genes included Pparg, Adipoq, Retn, Cfd and Cidec. Pparg and Adipoq down-regulation were confirmed by RT-PCR. PPAR-γ binding to its gene response element, adiponectin protein secretion and triglyceride accumulation were decreased by RTV. t10,c12-CLA in the presence of RTV decreased the expression of Ppargand Adipoq in microarray and RT-PCR. c9,t11-CLA increased PPAR-γ binding to its gene response element. Both isomers increased triglyceride storage in the presence of RTV. Conclusion: Ritonavir altered genes involved in adipocyte differentiation, lipid accumulation and glucose metabolism. Down-regulation of Pparg may be mediated by changes in Cepba and regulatory genes Pparg1c and Nr1h3.
Project description:The ability to obtain purified biliverdin IX (BVIX) isomers other than the commercially available BVIXα is limited due to the low yields obtained by the chemical coupled oxidation of heme. Chemical oxidation requires toxic chemicals, has very poor BVIX yields (<0.05%), and is not conducive to scalable production. Alternative approaches utilizing recombinant E. coli BL21 expressing a cyanobacterial heme oxygenase have been employed for the production BVIXα, but yields are limited by the rate of endogenous heme biosynthesis. Furthermore, the emerging roles of BVIXβ and BVIXδ in biology and their lack of commercial availability has led to a need for an efficient and scalable method with the flexibility to produce all three physiologically relevant BVIX isomers. Herein, we have taken advantage of an optimized non-pathogenic E. coli Nissle (EcN(T7)) strain that encodes an endogenous heme transporter and an integrated T7 polymerase gene. Protein production of the Pseudomonas aeruginosa BVIXβ and BVIXδ selective heme oxygenase (HemO) or its BVIXα producing mutant (HemOa) in the EcN(T7) strain provides a scalable method to obtain all three isomers, that is not limited by the rate of endogenous heme biosynthesis, due to the natural ability of EcN(T7) to transport extracellular heme. Additionally, we have optimized our previous LC-MS/MS protocol for semi-preparative separation and validation of the BVIX isomers. Utilizing this new methodology for scalable production and separation we have increased the yields of the BVIXβ and -δ isomers >300-fold when compared to the chemical oxidation of heme.
Project description:The ability to store information is believed to have been crucial for the origin and evolution of life; however, little is known about the genetic polymers relevant to abiogenesis. Nitrogen heterocycles (N-heterocycles) are plausible components of such polymers as they may have been readily available on early Earth and are the means by which the extant genetic macromolecules RNA and DNA store information. Here, we report the reactivity of numerous N-heterocycles in highly complex mixtures, which were generated using a Miller-Urey spark discharge apparatus with either a reducing or neutral atmosphere, to investigate how N-heterocycles are modified under plausible prebiotic conditions. High throughput mass spectrometry was used to identify N-heterocycle adducts. Additionally, tandem mass spectrometry and nuclear magnetic resonance spectroscopy were used to elucidate reaction pathways for select reactions. Remarkably, we found that the majority of N-heterocycles, including the canonical nucleobases, gain short carbonyl side chains in our complex mixtures via a Strecker-like synthesis or Michael addition. These types of N-heterocycle adducts are subunits of the proposed RNA precursor, peptide nucleic acids (PNAs). The ease with which these carbonylated heterocycles form under both reducing and neutral atmospheres is suggestive that PNAs could be prebiotically feasible on early Earth.
Project description:Elucidating the isomeric structure of free fatty acids (FAs) in biological samples is essential to comprehend their biological functions in various physiological and pathological processes. Herein, we report a novel approach of using peracetic acid (PAA) induced epoxidation coupled with mass spectrometry (MS) for localization of the C[double bond, length as m-dash]C bond in unsaturated FAs, which enables both quantification and spatial visualization of FA isomers from biological samples. Abundant diagnostic fragment ions indicative of the C[double bond, length as m-dash]C positions were produced upon fragmentation of the FA epoxides derived from either in-solution or on-tissue PAA epoxidation of free FAs. The performance of the proposed approach was evaluated by analysis of FAs in human cell lines as well as mapping the FA isomers from cancer tissue samples with MALDI-TOF/TOF-MS. Merits of the newly developed method include high sensitivity, simplicity, high reaction efficiency, and capability of spatial characterization of FA isomers in tissue samples.