Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed ...

Washington State University

In this study, R. opacus PD630, R. jostii RHA1, R. jostii RHA1 VanA, and their co-culture were employed to convert hydrothermal liquefaction aqueous waste (HTLAW) into lipids. After 11days, the COD reduction of algal-HTLAW reached 93.4% and 92.7% by R. jostii RHA1 and its mutant VanA, respectively. Woody-HTLAW promoted lipid accumulation of 0.43glipid/gcell dry weight in R. opacus PD630 cells. Additionally, the total number of chemicals in HTLAW decreased by over 1/3 after 7days of coculture, and 0.10g/L and 0.46g/L lipids were incrementally accumulated in the cellular mass during the fermentation of wood- and algal-HTLAW, respectively. The GC-MS data supported that different metabolism pathways were followed when these Rhodococci strains degraded algae- and woody-HTLAW. These results indicated promising potential of bioconversion of under-utilized carbon and toxic compounds in HTLAW into useful products by selected Rhodococci.

Biological conversion of the aqueous wastes from hydrothermal liquefaction of algae and pine wood by Rhodococci.

Nitrogen supply is critical to the synthesis of intracellular PHA in various bacteria. However, the specific role of the nitrogen in synthesizing PHA from benzoate, a lignin model compound use for the study of bacteria catabolism of aromatics, is still not clear. In this study, two culture conditions were maintained for Pseudomonas putida KT2440 to produce PHA using benzoate as a carbon source. Under nitrogen-limited and surplus conditions, the accumulation of PHA was to 37.3% and 0.25% of cell dry weight, respectively. A model fit to the kinetics of biomass growth and PHA accumulation showed good agreement with data. GC-MS and NMR showed that PHA contained six hydroxyl fatty acid monomers under nitrogen-limited conditions, while two monomers were identified under nitrogen surplus conditions. The average molecular weight of PHA increased after the nitrogen source was exhausted. These results provide a promising strategy for optimization of lignin to PHA yields.

Kinetic understanding of nitrogen supply condition on biosynthesis of polyhydroxyalkanoate from benzoate by Pseudomonas putida KT2440.

Biological routes for utilizing both carbohydrates and lignin are important to reach the ultimate goal of bioconversion of full carbon in biomass into biofuels and biochemicals. Recent biotechnology advances have shown promises toward facilitating biological transformation of lignin into lipids. Natural and engineered  strains (e.g.,  PD630 RHA1, and  RHA1 VanA) have been demonstrated to utilize lignin for lipid production, and co-culture of them can promote lipid production from lignin.

Discovery of potential pathways for biological conversion of poplar wood into lipids by co-fermentation of  strains.

Posttranslational modifications of protein cysteine thiols play a significant role in redox regulation and the pathogenesis of human diseases. Herein, we report the characterization of the cellular redox landscape in terms of quantitative, site-specific occupancies of both S-glutathionylation (SSG) and total reversible thiol oxidation (total oxidation) in RAW 264.7 macrophage cells under basal conditions. The occupancies of thiol modifications for ~4000 cysteine sites were quantified, revealing a mean site occupancy of 4.0% for SSG and 11.9% for total oxidation, respectively. Correlations between site occupancies and structural features such as pKa, relative residue surface accessibility, and hydrophobicity were observed. Proteome-wide site occupancy analysis revealed that the average occupancies of SSG and total oxidation in specific cellular compartments correlate well with the expected redox potentials of respective organelles in macrophages, consistent with the notion of redox compartmentalization. The lowest average occupancies were observed in more reducing organelles such as the mitochondria (non-membrane) and nucleus, while the highest average occupancies were found in more oxidizing organelles such as endoplasmic reticulum (ER) and lysosome. Furthermore, a pattern of subcellular susceptibility to redox changes was observed under oxidative stress induced by exposure to engineered metal oxide nanoparticles. Peroxisome, ER, and mitochondria (membrane) are the organelles which exhibit the most significant redox changes; while mitochondria (non-membrane) and Golgi were observed as the organelles being most resistant to oxidative stress. Finally, it was observed that Cys residues at enzymatic active sites generally had a higher level of occupancy compared to non-active Cys residues within the same proteins, suggesting site occupancy as a potential indicator of protein functional sites. The raw data are available via ProteomeXchange with identifier PXD019913.

Stochiometric quantification of the thiol redox proteome of macrophages reveals subcellular compartmentalization and susceptibility to oxidative perturbations.

The thiol redox proteome refers to all proteins whose cysteine thiols are subjected to various redox-dependent posttranslational modifications (PTMs) including glutathionylation (SSG), nitrosylation (SNO), sulfenylation (SOH), and sulfhydration (SSH). These modifications can impact various aspects of protein function such as activity, binding, conformation, localization, and interactions with other molecules. To identify novel redox proteins in signaling and regulation, it is highly desirable to have robust redox proteomics methods that can provide global, site-specific, and stoichiometric quantification of redox PTMs. Mass spectrometry (MS)-based redox proteomics has emerged as the primary platform for broad characterization of thiol PTMs in cells and tissues. Herein, we review recent advances in MS-based redox proteomics approaches for quantitative profiling of redox PTMs at physiological or oxidative stress conditions and highlight some recent applications. Considering the relative maturity of available methods, emphasis will be on two types of modifications: ) total oxidation (i.e., all reversible thiol modifications), the level of which represents the overall redox state, and ) glutathionylation, a major form of reversible thiol oxidation. We also discuss the significance of stoichiometric measurements of thiol PTMs as well as future perspectives toward a better understanding of cellular redox regulatory networks in cells and tissues.

Characterization of cellular oxidative stress response by stoichiometric redox proteomics.

Efficient utilization of all available carbons from lignocellulosic biomass is critical for economic efficiency of a bioconversion process to produce renewable bioproducts. However, the metabolic responses that enable Pseudomonas putida to utilize mixed carbon sources to generate reducing power and polyhydroxyalkanoate (PHA) remain unclear. Previous research has mainly focused on different fermentation strategies, including the sequential feeding of xylose as the growth stage substrate and octanoic acid as the PHA-producing substrate, feeding glycerol as the sole carbon substrate, and co-feeding of lignin and glucose. This study developed a new strategy-co-feeding glycerol and lignin derivatives such as benzoate, vanillin, and vanillic acid in Pseudomonas putida KT2440-for the first time, which simultaneously improved both cell biomass and PHA production.

Xiaolu Li

Bioproducts Sciences and Engineering Laboratory,
Department of Biological Systems Engineering,
Bioproducts Sciences and Engineering Laboratory, Department of Biological Systems Engineering

Washington State University

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Xiaolu Li

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Bioproducts Sciences and Engineering Laboratory,Department of Biological Systems Engineering,Bioproducts Sciences and Engineering Laboratory, Department of Biological Systems Engineering

Washington State University

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Bioproducts Sciences and Engineering Laboratory,
Department of Biological Systems Engineering,
Bioproducts Sciences and Engineering Laboratory, Department of Biological Systems Engineering