Constructing Human Proteoform Families Using Intact-Mass and Top-Down Proteomics with a Multi-Protease Global Post-Translational Modification Discovery Database.

Complex human biomolecular processes are made possible by the diversity of human proteoforms. Constructing proteoform families, groups of proteoforms derived from the same gene, is one way to represent this diversity. Comprehensive, high-confidence identification of human proteoforms remains a central challenge in mass spectrometry-based proteomics. We have previously reported a strategy for proteoform identification using intact-mass measurements, and we have since improved that strategy by mass calibration based on search results, the use of a global post-translational modification discovery database, and the integration of top-down proteomics results with intact-mass analysis. In the present study, we combine these strategies for enhanced proteoform identification in total cell lysate from the Jurkat human T lymphocyte cell line. We collected, processed, and integrated three types of proteomics data (NeuCode-labeled intact-mass, label-free top-down, and multi-protease bottom-up) to maximize the number of confident proteoform identifications. The integrated analysis revealed 5950 unique experimentally observed proteoforms, which were assembled into 848 proteoform families. Twenty percent of the observed proteoforms were confidently identified at a 3.9% false discovery rate, representing 1207 unique proteoforms derived from 484 genes.

Proteoform Suite: Software for Constructing, Quantifying, and Visualizing Proteoform Families.

We present an open-source, interactive program named Proteoform Suite that uses proteoform mass and intensity measurements from complex biological samples to identify and quantify proteoforms. It constructs families of proteoforms derived from the same gene, assesses proteoform function using gene ontology (GO) analysis, and enables visualization of quantified proteoform families and their changes. It is applied here to reveal systemic proteoform variations in the yeast response to salt stress.

Elucidating Escherichia coli Proteoform Families Using Intact-Mass Proteomics and a Global PTM Discovery Database.

A proteoform family is a group of related molecular forms of a protein (proteoforms) derived from the same gene. We have previously described a strategy to identify proteoforms and elucidate proteoform families in complex mixtures of intact proteins. The strategy is based upon measurements of two properties for each proteoform: (i) the accurate proteoform intact-mass, measured by liquid chromatography/mass spectrometry (LC-MS), and (ii) the number of lysine residues in each proteoform, determined using an isotopic labeling approach. These measured properties are then compared with those extracted from a catalog of theoretical proteoforms containing protein sequences and localized post-translational modifications (PTMs) for the organism under study. A match between the measured properties and those in the catalog constitutes an identification of the proteoform. In the present study, this strategy is extended by utilizing a global PTM discovery database and is applied to the widely studied model organism Escherichia coli, providing the most comprehensive elucidation of E. coli proteoforms and proteoform families to date.

Multiplexed Sequence-Specific Capture of Chromatin and Mass Spectrometric Discovery of Associated Proteins.

Comprehensive understanding of a gene's expression and regulation at the molecular level requires identification of all proteins interacting with the gene. HyCCAPP (Hybridization Capture of Chromatin Associated Proteins for Proteomics) is an approach that uses single-stranded DNA oligonucleotides to capture specific genomic sequences in cross-linked chromatin fragments and identify associated proteins by mass spectrometry. Previous studies have shown HyCCAPP to provide useful information on protein-DNA interactions, revealing the proteins associated with the GAL1-10 region in yeast. We present here a multiplexed version of HyCCAPP. Utilizing a toehold-mediated capture/release strategy, HyCCAPP is targeted to multiple genomic loci in parallel, and the protein binders at each locus are eluted in a programmable and selective fashion. Multiplexed HyCCAPP was applied to four genes (25S rDNA, ARX1, CTT1, and RPL30) in S. cerevisiae under normal and stressed conditions. Capture and release efficiencies and specificities were comparable to those obtained without multiplexing. Using mass spectrometry-based bottom-up proteomics, hundreds of proteins were discovered at each locus in each condition. Statistical analysis revealed 34-88 enriched proteins in each gene capture. Many of these proteins had expected functions, including DNA-related and ribosome biogenesis-associated activities. Multiplexed HyCCAPP provides a useful strategy for the identification of proteins interacting with specific chromatin regions.

Interaction, bioaccessibility and stability of bovine serum albumin-gamma-oryzanol complex: Spectroscopic and computational approaches.

The study explored the interaction mechanism between bovine serum albumin (BSA) and gamma-oryzanol (GO) by spectroscopic and computational approaches and the potential to enhance bioaccessibility and chemical stability of GO in the complex with BSA. Fluorescence spectroscopy showed that GO was bound to BSA with static quenching at a single binding site, being consistent with molecular docking results. Thermodynamic analysis and molecular dynamics simulation showed that electrostatic forces dominated interactions between BSA and GO. Besides, BSA-GO complex was more stable at pH 7.4 than at pH 2.0, with low root-mean-square deviation (2.57 Å vs 12.37 Å) and low binding energy (-424.23 kJ/mol at 277 K vs -188.55 kJ/mol at 277 K), but complex stability significantly decreased with increasing temperature. The bioaccessibility and stability of GO in the complex were significantly higher than those in water. This study provided theoretical support for developing proteins as delivery system for GO.

Improved catalytic properties of Candida antarctica lipase B immobilized on cetyl chloroformate-modified cellulose nanocrystals.

The catalytic activity of Candida antarctica lipase B (CALB) immobilized on modified cellulose nanocrystals (CNC) with different hydrophobicity was investigated using experimental and theoretical approaches. Firstly, the modified CNC were characterized by multi-spectroscopic methods, water contact angle, scanning electron microscopy and thermogravimetric analysis. Moderately hydrophobic CNC were found to be an optimal support for CALB immobilization. Secondly, model systems contained a CALB molecule and different numbers of modified CNC molecules (CALB@3CNC-C16, CALB@10CNC-C16 and CALB@15CNC-C16) were prepared for molecular dynamics (MD) simulation. Root-mean-square fluctuation values (0.61-2.61 Å) of lid region were relatively high in CALB@10CNC-C16, indicating that modified CNC with moderate hydrophobicity favored forming a lid-open conformation of CALB. Finally, the esterification of oleic acid catalyzed by the immobilized CALB showed higher conversion (54.68 %) than free CALB (12.98 %). Insights into modified CNC with tunable properties provided by this study may be a potential support for improving the catalytic performance of lipases.

Enhanced protein isoform characterization through long-read proteogenomics.

BACKGROUND: The detection of physiologically relevant protein isoforms encoded by the human genome is critical to biomedicine. Mass spectrometry (MS)-based proteomics is the preeminent method for protein detection, but isoform-resolved proteomic analysis relies on accurate reference databases that match the sample; neither a subset nor a superset database is ideal. Long-read RNA sequencing (e.g., PacBio or Oxford Nanopore) provides full-length transcripts which can be used to predict full-length protein isoforms. RESULTS: We describe here a long-read proteogenomics approach for integrating sample-matched long-read RNA-seq and MS-based proteomics data to enhance isoform characterization. We introduce a classification scheme for protein isoforms, discover novel protein isoforms, and present the first protein inference algorithm for the direct incorporation of long-read transcriptome data to enable detection of protein isoforms previously intractable to MS-based detection. We have released an open-source Nextflow pipeline that integrates long-read sequencing in a proteomic workflow for isoform-resolved analysis. CONCLUSIONS: Our work suggests that the incorporation of long-read sequencing and proteomic data can facilitate improved characterization of human protein isoform diversity. Our first-generation pipeline provides a strong foundation for future development of long-read proteogenomics and its adoption for both basic and translational research.

Aberrant miR-219-5p is correlated with TLR4 and serves as a novel biomarker in patients with multiple organ dysfunction syndrome caused by acute paraquat poisoning.

This study aimed to investigate the clinical significance of serum microRNA-219-5p (miR-219-5p) in patients with multiple organ dysfunction syndrome (MODS) caused by acute paraquat (PQ) poisoning, and its correlation with Toll-like Receptor 4 (TLR4). Luciferase reporter assay was used to investigate in vitro the correlation of miR-219-5p with TLR4. Serum miR-219-5p levels were evaluated by quantitative real-time polymerase chain reaction. Serum levels of TLR4, IL-1ß, and TNF-α were measured by Enzyme-linked immune sorbent assay (ELISA). ROC analysis was performed to assess the diagnostic significance, Kaplan-Meier survival curves and Cox regression analysis were used to evaluate the prognostic value of miR-219-5p in MODS patients. TLR4 was a target gene of miR-219-5p and was increased in MODS patients. Serum miR-219-5p level was decreased and negatively correlated with TLR4 level in MODS patients (r = -0.660, P < 0.001), which had important diagnostic value and negatively correlated with APACHE II score in MODS patients. The miR-219-5p expression was markedly associated with the WBC, ALT, AST, PaCO2, Lac, and APACHE II score. Non-survivals had more patients with low miR-219-5p expression. Patients with low miR-219-5p expression had shorter survival time. MiR-219-5p and APACHE II score were two independently prognostic factors for 28-day survival. MiR-219-5p was negatively correlated with, while TLR4 was positively correlated with the levels of IL-1ß and TNF-α. The serum miR-219-5p level may be a potential biomarker for acute PQ-induced MODS diagnosis and prognosis. Furthermore, miR-219-5p may be associated with the progression of MODS by regulating TLR4-related inflammatory response.

Silence of miR-32-5p promotes endothelial cell viability by targeting KLF2 and serves as a diagnostic biomarker of acute myocardial infarction.

BACKGROUND: MicroRNAs (miRNAs) have been investigated in various cardiovascular diseases. As a fatal disease, acute myocardial infarction (AMI) is a serious global health burden. The purpose of this study was to investigate the role of miR-32-5p in AMI patients and human umbilical vein endothelial cells (HUVECs) to explore novel diagnostic and therapeutic approaches for AMI. METHODS: A target prediction tool miRanda and the luciferase activity assay were used to confirm the interaction of miR-32-5p with Kruppel-like factor 2 (KLF2). Effect of miR-32-5p on HUVECs viability was examined using CCK-8 assay. Serum miR-32-5p expression was measured using quantitative Real-Time PCR, and its correlation with myocardial damage and endothelial injury markers and pro-inflammatory cytokines was assessed. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic value of miR-32-5p in AMI patients. RESULTS: miR-32-5p, as a direct regulator of KLF2, could suppress the cell proliferation of HUVECs. Serum miR-32-5p expression was elevated in AMI patients and positively correlated with the biomarker levels of myocardial damage and endothelial injury and pro-inflammatory cytokines. The area under the ROC curve for miR-32-5p was 0.949, indicating the relatively high diagnostic accuracy of miR-32-5p in AMI patients. CONCLUSION: The data of this study revealed that the increased serum miR-32-5p expression serves as a candidate diagnostic biomarker of AMI, and that miR-32-5p may be involved in the myocardial damage, endothelial injury and inflammatory responses of AMI by targeting KLF2, indicating the potential of miR-32-5p as a diagnostic biomarker and molecular target to improve the treatment of AMI.

Synthesis of Bi2WO6/Na-bentonite composites for photocatalytic oxidation of arsenic(iii) under simulated sunlight.

Novel Bi2WO6/bentonite (denoted as BWO/BENT) composites were prepared via a typical hydrothermal process and employed for the photocatalytic oxidation of arsenic(iii) (As(iii)). The properties of the prepared samples were characterized through X-ray diffraction, transmission and scanning electron microscopy, UV-visible diffuse reflectance spectroscopy, X-ray photoelectron spectroscopy, and photoluminescence spectroscopy. Effects of the BENT ratio on the As(iii) removal were explored under simulated sunlight, and the best photocatalytic effect was observed for the composite with BWO : BENT = 7 : 3 w/w. Compared with the pure BWO, the BWO/BENT composites exhibited an improved photocatalytic ability in the removal of As(iii), which was mainly ascribed to the enlarged specific surface area and the suppressed electron-hole recombination by the incorporated BENT. Furthermore, photo-generated holes (h+) and superoxide radicals ·O2 - were confirmed to be the major contributors to the oxidation of As(iii), and an associated mechanism of photocatalytic oxidation of As(iii) over BWO/BENT composites was proposed.

Correction: Synthesis of Bi2WO6/Na-bentonite composites for photocatalytic oxidation of arsenic(iii) under simulated sunlight.

[This corrects the article DOI: 10.1039/C9RA06181A.].