Rat Bronchoalveolar Lavage Proteome Changes Following E-cigarette Aerosol Exposures

Supplemental material to e-cigarette BAL proteomics manuscript at AJP Lung</p

Detection and Imaging of Zinc Secretion from Pancreatic β-Cells Using a New Fluorescent Zinc Indicator

A novel Zn2+-selective visible wavelength fluoroionophore (FluoZin-3, 9) was synthesized. The chelating portion of the molecule resembles known EGTA-based Ca2+-selective fluoroionophores, except that one of the N-acetic acid moieties has been deleted in 9. FluoZin-3 is virtually non-fluorescent in the absence of Zn2+, and exhibits a several hundred-fold fluorescence increase upon saturation with Zn2+(∼100 nM), with a Kd = 15 ± 2 nM. A 1:1 binding stoichiometry of 9:Zn2+ was determined, and the fluorescence of the complex is pH-independent at pH > 6. FluoZin-3 was used to monitor Zn2+ that was co-secreted with insulin from pancreatic β-cells by exocytosis following stimulation with glucose. The total Zn2+ concentration near the cells reached 600 nM, and Zn2+ was detectable at least 15 μm away from secreting cells. Heterogeneity in secretion among cells was indicated in that some cells in a cluster did not release Zn2+. Also, within secreting cells some regions of the cell membrane gave rise to secretion while others did not, suggesting active zones of secretion on the cell surface

A Sweet H₂S/H₂O₂ Dual Release System and Specific Protein S‑Persulfidation Mediated by Thioglucose/Glucose Oxidase

H2S and H2O2 are two redox regulating molecules that play important roles in many physiological and pathological processes. While each of them has distinct biosynthetic pathways and signaling mechanisms, the crosstalk between these two species is also known to cause critical biological responses such as protein S-persulfidation. So far, many chemical tools for the studies of H2S and H2O2 have been developed, such as the donors and sensors for H2S and H2O2. However, these tools are normally targeting single species (e.g., only H2S or only H2O2). As such, the crosstalk and synergetic effects between H2S and H2O2 have hardly been studied with those tools. In this work, we report a unique H2S/H2O2 dual donor system by employing 1-thio-β-d-glucose and glucose oxidase (GOx) as the substrates. This enzymatic system can simultaneously produce H2S and H2O2 in a slow and controllable fashion, without generating any bio-unfriendly byproducts. This system was demonstrated to cause efficient S-persulfidation on proteins. In addition, we expanded the system to thiolactose and thioglucose-disulfide; therefore, additional factors (β-galactosidase and cellular reductants) could be introduced to further control the release of H2S/H2O2. This dual release system should be useful for future research on H2S and H2O2

In-Source Fragmentation and the Sources of Partially Tryptic Peptides in Shotgun Proteomics

Partially tryptic peptides are often identified in shotgun proteomics using trypsin as the proteolytic enzyme; however, their sources have been controversial. Herein, we investigate the impact of in-source fragmentation on shotgun proteomics profiling of three biological samples: a standard protein mixture, a mouse brain tissue homogenate, and mouse plasma. Because the in-source fragments of peptide ions have the same LC elution time as their parental peptides, partially tryptic peptide ions from in-source fragmentation can be distinguished from other partially tryptic peptides based on their elution time differences from those computationally predicted data. The percentage of partially tryptic peptide identifications resulting from in-source fragmentation in a standard protein digest was observed to be ∼60%. In more complex mouse brain or plasma samples, in-source fragmentation contributed to a lesser degree of 1–3% of all identified peptides due to the limited dynamic range of LC–MS/MS measurements. The other major source of partially tryptic peptides in complex biological samples is presumably proteolytic cleavage by endogenous proteases in the samples. Our work also provides a method to identify such proteolytic-derived partially tryptic peptides due to endogenous proteases in the samples by removing in-source fragmentation artifacts from the identified peptides

MOESM1 of Biodegradation of alkaline lignin by Bacillus ligniniphilus L1

Additional file 1. Up regulation of expressed protein with lignin as carbon source

MOESM2 of Biodegradation of alkaline lignin by Bacillus ligniniphilus L1

Additional file 2. Down regulation of expressed protein with lignin as carbon source

Controllable Cycloadditions between 2<i>H</i>‑(Thio)pyran-2-(thi)ones and Strained Alkynes: A Click-and-Release Strategy for COS/H₂S Generation

In this work, we carried out computational studies to predict the cycloaddition efficiency of strained alkynes with 2H-pyran-2-one and its three sulfur-containing analogues: 2H-pyran-2-thione, 2H-thiopyran-2-one, and 2H-thiopyran-2-thione. It was predicted that the decreased aromaticity of the substrate would yield higher reactivity. Experimental studies confirmed the calculation results, and 2H-pyan-2-thiones were found to be the most reactive substrates. This reaction proceeded effectively in aqueous buffers and in cellular environments. It also produced COS as the byproduct, which could be converted into hydrogen sulfide (H2S) in the presence of carbonate anhydrase. This click-and-release approach may serve as a unique way to deliver COS/H2S to specific locations

In-Source Fragmentation and the Sources of Partially Tryptic Peptides in Shotgun Proteomics

Partially tryptic peptides are often identified in shotgun proteomics using trypsin as the proteolytic enzyme; however, their sources have been controversial. Herein, we investigate the impact of in-source fragmentation on shotgun proteomics profiling of three biological samples: a standard protein mixture, a mouse brain tissue homogenate, and mouse plasma. Because the in-source fragments of peptide ions have the same LC elution time as their parental peptides, partially tryptic peptide ions from in-source fragmentation can be distinguished from other partially tryptic peptides based on their elution time differences from those computationally predicted data. The percentage of partially tryptic peptide identifications resulting from in-source fragmentation in a standard protein digest was observed to be ∼60%. In more complex mouse brain or plasma samples, in-source fragmentation contributed to a lesser degree of 1–3% of all identified peptides due to the limited dynamic range of LC–MS/MS measurements. The other major source of partially tryptic peptides in complex biological samples is presumably proteolytic cleavage by endogenous proteases in the samples. Our work also provides a method to identify such proteolytic-derived partially tryptic peptides due to endogenous proteases in the samples by removing in-source fragmentation artifacts from the identified peptides

Development of Multiplexed Immuno-N-Terminomics to Reveal the Landscape of Proteolytic Processing in Early Embryogenesis of <i>Drosophila melanogaster</i>

Protein expression levels are regulated through both translation and degradation mechanisms. Levels of degradation intermediates, that is, partially degraded proteins, cannot be distinguished from those of intact proteins by global proteomics analysis, which quantify total protein abundance levels. This study aimed to develop a tool for assessing the aspects of degradation regulation via proteolytic processing through a new multiplexed N-terminomics method involving selective isobaric labeling of protein N-termini and immunoaffinity capture of the labeled N-terminal peptides. Our method allows for not only identification of proteolytic cleavage sites, but also highly multiplexed quantification of proteolytic processing. We profiled a number of potential cleavage sites by signal peptidase and provided experimental confirmation of predicted cleavage sites of signal peptide. Furthermore, the present method uniquely represents the landscape of proteomic proteolytic processing rate during early embryogenesis in Drosophila melanogaster, revealing the underlying mechanism of stringent decay regulation of zygotically expressed proteins during early stages of embryogenesis

Phosphoprotein Isotope-Coded Solid-Phase Tag Approach for Enrichment and Quantitative Analysis of Phosphopeptides from Complex Mixtures

Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory,, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated β-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography−tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line