Aqueous humour protein dysregulation in Asian eyes with primary open angle glaucoma.

The pathophysiology of Primary Open Angle Glaucoma (POAG) remains poorly understood. Through proteomic analysis of aqueous humour (AH) from POAG patients, we aim to identify changes in protein composition of these samples compared to control samples. High resolution mass spectrometry-based TMT6plex quantitative proteomics analysis is performed on AH samples collected from POAG patients, and compared against a control group of patients with cataracts. Data are available via ProteomeXchange with identifier PXD033153. 1589 proteins were quantified from the aqueous samples using Proteome Discoverer version 2.2 software. Among these proteins, 210 were identified as unique master proteins. The proteins which were up or down-regulated by ±3 fold-change were considered significant. Human neuroblastoma full-length cDNA clone CS0DD006YL02 was significantly upregulated in patients with severe POAG on >2 medications, while actin, cytoplasmic 1, V2-7 protein (fragment), immunoglobulin-like polypeptide 1 and phosphatidylethanolamine-binding protein 4 were only present in these patients with severe POAG on >2 medications. Beta-crystallin B1 and B2, Gamma-crystallin C, D and S were significantly downregulated in the severe POAG ≤2 glaucoma medications group. Beta-crystallin B2, Gamma-crystallin D and GCT-A9 light chain variable region (fragment) were significantly downregulated in the non-severe POAG group. Actin, cytoplasmic 1 was significantly upregulated in subjects with severe POAG who required more than 2 glaucoma medications. Crystallins (Beta-crystallin B1 and B2, Gamma-crystallin C, D and S) were significantly downregulated in subjects with severe POAG who required less than 2 glaucoma medications.

Resident macrophages restrain pathological adipose tissue remodeling and protect vascular integrity in obese mice.

Tissue-resident macrophages in white adipose tissue (WAT) dynamically adapt to the metabolic changes of their microenvironment that are often induced by excess energy intake. Currently, the exact contribution of these macrophages in obesity-driven WAT remodeling remains controversial. Here, using a transgenic CD169-DTR mouse strain, we provide new insights into the interplay between CD169+ adipose tissue macrophages (ATMs) and their surrounding WAT microenvironment. Using targeted in vivo ATM ablation followed by transcriptional and metabolic WAT profiling, we found that ATMs protect WAT from the excessive pathological remodeling that occurs during obesity. As obesity progresses, ATMs control not only vascular integrity, adipocyte function, and lipid and metabolic derangements but also extracellular matrix accumulation and resultant fibrosis in the WAT. The protective role of ATMs during obesity-driven WAT dysfunction supports the notion that ATMs represent friends, rather than foes, as has previously assumed.

Serum albumin cysteine trioxidation is a potential oxidative stress biomarker of type 2 diabetes mellitus.

Metabolic disorders in T2DM generate multiple sources of free radicals and oxidative stress that accelerate nonenzymatic degenerative protein modifications (DPMs) such as protein oxidation, disrupt redox signaling and physiological function, and remain a major risk factor for clinical diabetic vascular complications. In order to identify potential oxidative biomarkers in the blood plasma of patients with T2DM, we used LC-MS/MS-based proteomics to profile plasma samples from patients with T2DM and healthy controls. The results showed that human serum albumin (HSA) is damaged by irreversible cysteine trioxidation, which can be a potential oxidative stress biomarker for the early diagnosis of T2DM. The quantitative detection of site-specific thiol trioxidation is technically challenging; thus, we developed a sensitive and selective LC-MS/MS workflow that has been used to discover and quantify three unique thiol-trioxidized HSA peptides, ALVLIAFAQYLQQC(SO3H)PFEDHVK (m/z 1241.13), YIC(SO3H)ENQDSISSK (m/z 717.80) and RPC(SO3H)FSALEVDETYVPK (m/z 951.45), in 16 individual samples of healthy controls (n = 8) and individuals with diabetes (n = 8). Targeted quantitative analysis using multiple reaction monitoring mass spectrometry revealed impairment of the peptides with m/z 1241.13, m/z 717.80 and m/z 951.45, with significance (P < 0.02, P < 0.002 and P < 0.03), in individuals with diabetes. The results demonstrated that a set of three HSA thiol-trioxidized peptides, which are irreversibly oxidatively damaged in HSA in the plasma of patients with T2DM, can be important indicators and potential biomarkers of oxidative stress in T2DM.

Integrated Transcriptomics, Metabolomics, and Lipidomics Profiling in Rat Lung, Blood, and Serum for Assessment of Laser Printer-Emitted Nanoparticle Inhalation Exposure-Induced Disease Risks.

Laser printer-emitted nanoparticles (PEPs) generated from toners during printing represent one of the most common types of life cycle released particulate matter from nano-enabled products. Toxicological assessment of PEPs is therefore important for occupational and consumer health protection. Our group recently reported exposure to PEPs induces adverse cardiovascular responses including hypertension and arrythmia via monitoring left ventricular pressure and electrocardiogram in rats. This study employed genome-wide mRNA and miRNA profiling in rat lung and blood integrated with metabolomics and lipidomics profiling in rat serum to identify biomarkers for assessing PEPs-induced disease risks. Whole-body inhalation of PEPs perturbed transcriptional activities associated with cardiovascular dysfunction, metabolic syndrome, and neural disorders at every observed time point in both rat lung and blood during the 21 days of exposure. Furthermore, the systematic analysis revealed PEPs-induced transcriptomic changes linking to other disease risks in rats, including diabetes, congenital defects, auto-recessive disorders, physical deformation, and carcinogenesis. The results were also confirmed with global metabolomics profiling in rat serum. Among the validated metabolites and lipids, linoleic acid, arachidonic acid, docosahexanoic acid, and histidine showed significant variation in PEPs-exposed rat serum. Overall, the identified PEPs-induced dysregulated genes, molecular pathways and functions, and miRNA-mediated transcriptional activities provide important insights into the disease mechanisms. The discovered important mRNAs, miRNAs, lipids and metabolites may serve as candidate biomarkers for future occupational and medical surveillance studies. To the best of our knowledge, this is the first study systematically integrating in vivo, transcriptomics, metabolomics, and lipidomics to assess PEPs inhalation exposure-induced disease risks using a rat model.

Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo.

The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major contributor to wound healing impairments. Proteolytic peptide patterns could therefore correlate and "report" healing activity and infection. This work describes a proof of principle delineating a strategy by which peptides from a selected protein, human thrombin, are detected and attributed to proteolytic actions. With a particular focus on thrombin-derived C-terminal peptides (TCP), we show that distinct peptide patterns are generated in vitro by the human S1 peptidases human neutrophil elastase and cathepsin G, and the bacterial M4 peptidases Pseudomonas aeruginosa elastase and Staphylococcus aureus aureolysin, respectively. Corresponding peptide sequences were identified in wound fluids from acute and non-healing ulcers, and notably, one peptide, FYT21 (FYTHVFRLKKWIQKVIDQFGE), was only present in wound fluid from non-healing ulcers colonized by P. aeruginosa and S. aureus. Our result is a proof of principle pointing at the possibility of defining peptide biomarkers reporting distinct proteolytic activities, of potential implication for improved diagnosis of wound healing and infection.

Recent advances in mass spectrometric analysis of protein deamidation.

Protein deamidation has been proposed to represent a "molecular clock" that progressively disrupts protein structure and function in human degenerative diseases and natural aging. Importantly, this spontaneous process can also modify therapeutic proteins by altering their purity, stability, bioactivity, and antigenicity during drug synthesis and storage. Deamidation occurs non-enzymatically in vivo, but can also take place spontaneously in vitro, hence artificial deamidation during proteomic sample preparation can hamper efforts to identify and quantify endogenous deamidation of complex proteomes. To overcome this, mass spectrometry (MS) can be used to conduct rigorous site-specific characterization of protein deamidation due to the high sensitivity, speed, and specificity offered by this technique. This article reviews recent progress in MS analysis of protein deamidation and discusses the strengths and limitations of common "top-down" and "bottom-up" approaches. Recent advances in sample preparation methods, chromatographic separation, MS technology, and data processing have for the first time enabled the accurate and reliable characterization of protein modifications in complex biological samples, yielding important new data on how deamidation occurs across the entire proteome of human cells and tissues. These technological advances will lead to a better understanding of how deamidation contributes to the pathology of biological aging and major degenerative diseases. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:677-692, 2017.

Dementia-linked amyloidosis is associated with brain protein deamidation as revealed by proteomic profiling of human brain tissues.

BACKGROUND: Aggregation of malformed proteins is a key feature of many neurodegenerative diseases, but the mechanisms that drive proteinopathy in the brain are poorly understood. We aimed to characterize aggregated proteins in human brain tissues affected by dementia. RESULTS: To characterize amyloidal plaque purified from post-mortem brain tissue of dementia patient, we applied ultracentrifugation-electrostatic repulsion hydrophilic interaction chromatography (UC-ERLIC) coupled mass spectrometry-based proteomics technologies. Proteomics profiling of both soluble and aggregated amyloidal plaque demonstrated significant enrichment and deamidation of S100A9, ferritin, hemoglobin subunits, creatine kinase and collagen protein among the aggregated brain proteins. Amyloidal plaques were enriched in the deamidated variant of protein S100A9, and structural analysis indicated that both the low- and high-affinity calcium binding motifs of S100A9 were deamidated exclusively in the aggregated fraction, suggesting altered charge state and function of this protein in brain tissues affected by dementia. The multiple deamidated residues of S100A9 predicts introduction of negative charge that alter Ca(++) binding, suggesting increased capacity to form pathological aggregates in the brain. CONCLUSION: UC-coupled proteomics revealed that brain amyloidal plaques are enriched in deamidated proteins, and suggested that altered charge state and calcium-binding capacity of S100A9 may enhance protein aggregation and promote neurodegeneration in the human brain.

Alishewanella solinquinati sp. nov., isolated from soil contaminated with textile dyes.

A novel Gram-negative, motile, rod-shaped, facultative anaerobic bacterial strain, KMK6(T), was isolated from soil contaminated with textile dyes from an industrial estate located at Ichalkaranji, Maharashtra, India, and its taxonomical position was established by using a polyphasic approach. The major cellular fatty acids included C17:1ω8c, summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH), C17:0, C16:0, and C18:1ω7c. The DNA G+C content of strain KMK6(T) was 48.8 mol %. 16S rRNA gene sequence analysis confirmed its placement in the genus Alishewanella, and exhibited sequence similarity levels of below 97 % to the type strains of validly published Alishewanella species. On the basis of genotypic and phenotypic evidence, strains KMK6(T) is considered to be a novel species of the genus Alishewanella, for which we propose that strain KMK6(T) (=NCIM 5295(T) =BCRC 17848(T)) is assigned to a novel species, Alishewanella solinquinati sp. nov.

Elucidating the temporal dynamics of chromatin-associated protein release upon DNA digestion by quantitative proteomic approach.

Chromatin is a highly dynamic well organized nucleoprotein complex of DNA and proteins that controls DNA-dependent processes such as transcription, replication, repair and many others. Chromatin structure is regulated by various chromatin associated proteins, post-translational modifications of histones and DNA methylation, but a complete picture of structural changes in chromatin architecture is unclear due to the lack of comprehensive data of chromatin-associated proteins and their bindings to different chromatin regions. This study temporally released chromatin-associated proteins by DNase I and MNase treatment and profiled them by exponentially modified protein abundance index (emPAI) based label-free quantitative proteomics. We identified 694 high confidence proteins, with 160 known chromatin-associated proteins. Identified proteins were functionally classified into histones, non-histones involved in architectural maintenance, proteins involved in DNA replication and repair, transcription machinery, transcription regulation, other chromatin proteins, cell cycle proteins and several novel proteins. Numerous proteins presumed to be chromatin associated were identified and their chromatin interactions were explored. The comprehensive differential chromatin bound proteome might expand our knowledge of the proteins that were associated with different chromatin regions, which could be very useful in elucidating chromatin biology.