Cheng-Kang Chiang

Altered intestinal microbiota–host mitochondria crosstalk in new onset Crohn’s disease

Intestinal microbial dysbiosis is associated with Crohns disease (CD). However, the mechanisms leading to the chronic mucosal inflammation that characterizes this disease remain unclear. In this report, we use systems-level approaches to study the interactions between the gut microbiota and host in new-onset paediatric patients to evaluate causality and mechanisms of disease. We report an altered host proteome in CD patients indicative of impaired mitochondrial functions. In particular, mitochondrial proteins implicated in H2S detoxification are downregulated, while the relative abundance of H2S microbial producers is increased. Network correlation analysis reveals that Atopobium parvulum controls the central hub of H2S producers. A. parvulum induces pancolitis in colitis-susceptible interleukin-10-deficient mice and this phenotype requires the presence of the intestinal microbiota. Administrating the H2S scavenger bismuth mitigates A. parvulum-induced colitis in vivo. This study reveals that host–microbiota interactions are disturbed in CD and thus provides mechanistic insights into CD pathogenesis.

MetaPro-IQ: a universal metaproteomic approach to studying human and mouse gut microbiota

BackgroundThe gut microbiota has been shown to be closely associated with human health and disease. While next-generation sequencing can be readily used to profile the microbiota taxonomy and metabolic potential, metaproteomics is better suited for deciphering microbial biological activities. However, the application of gut metaproteomics has largely been limited due to the low efficiency of protein identification. Thus, a high-performance and easy-to-implement gut metaproteomic approach is required.ResultsIn this study, we developed a high-performance and universal workflow for gut metaproteome identification and quantification (named MetaPro-IQ) by using the close-to-complete human or mouse gut microbial gene catalog as database and an iterative database search strategy. An average of 38 and 33xa0% of the acquired tandem mass spectrometry (MS) spectra was confidently identified for the studied mouse stool and human mucosal-luminal interface samples, respectively. In total, we accurately quantified 30,749 protein groups for the mouse metaproteome and 19,011 protein groups for the human metaproteome. Moreover, the MetaPro-IQ approach enabled comparable identifications with the matched metagenome database search strategy that is widely used but needs prior metagenomic sequencing. The response of gut microbiota to high-fat diet in mice was then assessed, which showed distinct metaproteome patterns for high-fat-fed mice and identified 849 proteins as significant responders to high-fat feeding in comparison to low-fat feeding.ConclusionsWe present MetaPro-IQ, a metaproteomic approach for highly efficient intestinal microbial protein identification and quantification, which functions as a universal workflow for metaproteomic studies, and will thus facilitate the application of metaproteomics for better understanding the functions of gut microbiota in health and disease.

The proteomic landscape of the suprachiasmatic nucleus clock reveals large-scale coordination of key biological processes.

The suprachiasmatic nucleus (SCN) acts as the central clock to coordinate circadian oscillations in mammalian behavior, physiology and gene expression. Despite our knowledge of the circadian transcriptome of the SCN, how it impacts genome-wide protein expression is not well understood. Here, we interrogated the murine SCN proteome across the circadian cycle using SILAC-based quantitative mass spectrometry. Of the 2112 proteins that were accurately quantified, 20% (421 proteins) displayed a time-of-day-dependent expression profile. Within this time-of-day proteome, 11% (48 proteins) were further defined as circadian based on a sinusoidal expression pattern with a ∼24 h period. Nine circadianly expressed proteins exhibited 24 h rhythms at the transcript level, with an average time lag that exceeded 8 h. A substantial proportion of the time-of-day proteome exhibited abrupt fluctuations at the anticipated light-to-dark and dark-to-light transitions, and was enriched for proteins involved in several key biological pathways, most notably, mitochondrial oxidative phosphorylation. Additionally, predicted targets of miR-133ab were enriched in specific hierarchical clusters and were inversely correlated with miR133ab expression in the SCN. These insights into the proteomic landscape of the SCN will facilitate a more integrative understanding of cellular control within the SCN clock.

From cells to peptides: "one-stop" integrated proteomic processing using amphipols.

In proteomics, detergents and chaotropes are indispensable for proteome analysis, not only for protein extraction, but also for protein digestion. To increase the protein extraction efficiency, detergents are usually added in the lysis buffer to extract membrane proteins out of membrane structure and to maintain protein in solutions. In general, these detergents need to be removed prior to protein digestion, usually by precipitation or ultrafiltration. Digestion often takes place in the presence of chaotropic reagents, such as urea, which often need to be removed prior to mass spectrometry. The addition and removal of detergents and chaotropes require multiple steps that are time-consuming and can cause sample losses. Amphipols (APols) are a different class of detergents that have physical and solubilization properties that are distinct from conventional detergents. They have primarily been used in protein structure analysis for membrane protein trapping and stabilization. Here, we demonstrate a simple and rapid protocol for total and membrane proteome preparation using APols. We demonstrate that APols added for cell lysis help maintain the proteome in solution, are compatible with protein digestion using trypsin, and can readily be removed prior to mass spectrometry by a one-step acidification and centrifugation. This protocol is much faster, can be performed in a single tube, and can readily replace the conventional detergent/chaotrope approaches for total and membrane proteome analysis.

Fine Tuning of Proteomic Technologies to Improve Biological Findings: Advancements in 2011–2013

■ CONTENTS Sample Preparation 177 Protein Extraction 177 Secreted Proteins 177 Exosomes 177 Membrane Proteins 178 Protein Stabilization 178 Miniturizing and Automating Sample Preparation 178 Protein Digestion 178 Multiple Enzymes 178 Enzyme Immobilization 178 Decreasing Sample Complexity 178 Serum/Plasma Strategies to Improve Dynamic Range 178 Depletion Strategies 178 Enrichment Strategies 179 Enriching Post-Translationally Modified Proteomes 180 Phosphopeptide Enrichment 180 Glycopeptide Enrichment 180 Enrichment of Other PTMs 181 Methods to Improve Coverage 181 Filtered Aided Sample Preparation (FASP) 181 SDS Spin Columns 181 Detergent Clean-up Methods for MS-Deleterious Agents 182 Integrated Approaches 182 Monolithic Columns 182 Quantitative Proteomics 182 Gel Staining 182 Label-Free Quantitation 182 Metabolic Labeling/SILAC 183 Chemical Labeling 183 Targeted Quantitative Proteomics 183 Selected Reaction Monitoring 183 Characterizing Post-Translationally Modified Proteomes by Mass Spectrometry 184 Protein Interactions 185 Technologies 185 Methods 185 Applications 186 Bioinformatics 186 Proteomic Analyses/Bioinformatics 187 Databases 187 Search Engines 187 Software 188 Label-Free Software 188 Extended Analysis 188 From Proteomic to Biological Applications 188 Profiling Disease States 188 From Genes to Proteins 188 Biomarker Development 189 Identification of Novel Biomarkers 189 Applying Proteomics to Known Biomarkers 189 Conclusions 190 Author Information 190 Corresponding Author 190 Notes 190 Biographies 190 Acknowledgments 190 References 190

Proteomic analysis of ascending colon biopsies from a paediatric inflammatory bowel disease inception cohort identifies protein biomarkers that differentiate Crohn's disease from UC.

Objective Accurate differentiation between Crohns disease (CD) and UC is important to ensure early and appropriate therapeutic intervention. We sought to identify proteins that enable differentiation between CD and UC in children with new onset IBD. Design Mucosal biopsies were obtained from children undergoing baseline diagnostic endoscopy prior to therapeutic interventions. Using a super-stable isotope labeling with amino acids in cell culture (SILAC)-based approach, the proteomes of 99 paediatric control and biopsies of patients with CD and UC were compared. Multivariate analysis of a subset of these (n=50) was applied to identify novel biomarkers, which were validated in a second subset (n=49). Results In the discovery cohort, a panel of five proteins was sufficient to distinguish control from IBD-affected tissue biopsies with an AUC of 1.0 (95% CI 0.99 to 1.0); a second panel of 12 proteins segregated inflamed CD from UC within an AUC of 0.95 (95% CI 0.86 to 1.0). Application of the two panels to the validation cohort resulted in accurate classification of 95.9% (IBD from control) and 80% (CD from UC) of patients. 116 proteins were identified to have correlation with the severity of disease, four of which were components of the two panels, including visfatin and metallothionein-2. Conclusions This study has identified two panels of candidate biomarkers for the diagnosis of IBD and the differentiation of IBD subtypes to guide appropriate therapeutic interventions in paediatric patients.

GRK2 Fine-Tunes Circadian Clock Speed and Entrainment via Transcriptional and Post-translational Control of PERIOD Proteins.

The pacemaker properties of the suprachiasmatic nucleus (SCN) circadian clock are shaped by mechanisms that influence the expression and behavior of clock proteins. Here, we reveal that G-protein-coupled receptor kinase 2 (GRK2) modulates the period, amplitude, and entrainment characteristics of the SCN. Grk2-deficient mice show phase-dependent alterations in light-induced entrainment, slower recovery from jetlag, and longer behavioral rhythms. Grk2 ablation perturbs intrinsic rhythmic properties of the SCN, increasing amplitude and decreasing period. At the cellular level, GRK2 suppresses the transcription of the mPeriod1 gene and the trafficking of PERIOD1 and PERIOD2 proteins to the nucleus. Moreover, GRK2 can physically interact with PERIOD1/2 and promote PERIOD2 phosphorylation at Ser545, effects that may underlie its ability to regulate PERIOD1/2 trafficking. Together, our findings identify GRK2 as an important modulator of circadian clock speed, amplitude, and entrainment by controlling PERIOD at the transcriptional and post-translational levels.

Detecting protein-protein interactions/complex components using mass spectrometry coupled techniques.

Proteins play important roles in biochemical processes. Most biological functions are realized through protein-protein interactions (PPI). Co-immunoprecipitation is the most straightforward method to detect PPI. With the development of modern mass spectrometry (MS), throughput, sensitivity, and confidence for the detection of PPI can be readily achieved by scaling up traditional antibody-based strategies. Herein, we describe a typical workflow for general PPI detection using mass spectrometry coupled techniques, covering from Co-immunoprecipitation (Co-IP), to gel display, in-gel digestion, liquid chromatography mass spectrometry (LC-MS) analysis, as well as result interpretation and statistic filtering. This protocol provides an overview of the technique as well as practical tips.

Phosphoproteome Profiling Reveals Circadian Clock Regulation of Posttranslational Modifications in the Murine Hippocampus

The circadian clock is an endogenous oscillator that drives daily rhythms in physiology, behavior, and gene expression. The underlying mechanisms of circadian timekeeping are cell-autonomous and involve oscillatory expression of core clock genes that is driven by interconnecting transcription–translation feedback loops (TTFLs). Circadian clock TTFLs are further regulated by posttranslational modifications, in particular, phosphorylation. The hippocampus plays an important role in spatial memory and the conversion of short- to long-term memory. Several studies have reported the presence of a peripheral oscillator in the hippocampus and have highlighted the importance of circadian regulation in memory formation. Given the general importance of phosphorylation in circadian clock regulation, we performed global quantitative proteome and phosphoproteome analyses of the murine hippocampus across the circadian cycle, applying spiked-in labeled reference and high accuracy mass spectrometry (MS). Of the 3,052 proteins and 2,868 phosphosites on 1,368 proteins that were accurately quantified, 1.7% of proteins and 5.2% of phosphorylation events exhibited time-of-day-dependent expression profiles. The majority of circadian phosphopeptides displayed abrupt fluctuations at mid-to-late day without underlying rhythms of protein abundance. Bioinformatic analysis of cyclic phosphorylation events revealed their diverse distribution in different biological pathways, most notably, cytoskeletal organization and neuronal morphogenesis. This study provides the first large-scale, quantitative MS analysis of the circadian phosphoproteome and proteome of the murine hippocampus and highlights the significance of rhythmic regulation at the posttranslational level in this peripheral oscillator. In addition to providing molecular insights into the hippocampal circadian clock, our results will assist in the understanding of genetic factors that underlie rhythms-associated pathological states of the hippocampus.

Mucosal–luminal interface proteomics reveals biomarkers of pediatric inflammatory bowel disease-associated colitis

OBJECTIVE: Improved biomarkers are an unmet clinical need for suspected inflammatory bowel disease (IBD). Need is greatest for children, since current biomarkers suffers from low specificity, particularly in this population; thus, invasive testing methods, with the accompanying risk of complications, are necessary. Additionally, current biomarkers do not delineate disease extent assessment for ulcerative colitis (UC), a factor involved in therapeutic decisions. METHODS: Intestinal mucosal‐luminal interface (MLI) aspirates from the ascending colon (AC) and descending colon (DC) were collected during diagnostic colonoscopy from treatment‐naïve children. The MLI proteomes of 18 non‐IBD and 42 IBD patients were analyzed by liquid chromatography mass spectrometry. Analyses of proteomic data generated protein panels distinguishing IBD from non‐IBD and pancolitis from non‐pancolitis (UC disease extent). Select protein biomarkers were evaluated in stool samples by enzyme‐linked immunosorbent assay (n = 24). RESULTS: A panel of four proteins discriminated active IBD from non‐IBD (discovery cohort) with a sensitivity of 0.954 (95% confidence interval (CI): 0.772–0.999) and >0.999 (95% CI: 0.824–1.00) for the AC and DC, respectively, and a specificity of >0.999 (AC, 95% CI: 0.815–1.00; DC, 95% CI:0.692–1.00) for both the AC and DC. A separate panel of four proteins distinguished pancolitis from non‐pancolitis in UC patients with sensitivity >0.999 (95% CI: 0.590–1.00) and specificity >0.999 (95% CI: 0.715–1.00). Catalase (p < 0.0001) and LTA4H (p = 0.0002) were elevated in IBD stool samples compared to non‐IBD stool samples. CONCLUSION: This study identified panels of proteins that have significantly different expression levels and contribute to accurate IBD diagnosis and disease extent characterization in children with UC. Biomarkers identified from the MLI demonstrate transferable results in stool samples.