Comprehensive profiling of the human fecal proteome from IBD patients with DIA-MS enables evaluation of disease-relevant proteins.

PURPOSE: Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), is characterized by chronic gastrointestinal inflammation. A high unmet need exists for noninvasive biomarkers in IBD to monitor changes in disease activity and guide treatment decisions. Stool is an easily accessed, disease proximal matrix in IBD, however the composition of the IBD fecal proteome remains poorly characterized. EXPERIMENTAL DESIGN: A data-independent acquisition LC-MS/MS approach was used to profile the human fecal proteome in two independent cohorts (Cohort 1: healthy n = 5, UC n = 5, CD n = 5, Cohort 2: healthy n = 20, UC n = 10, and CD n = 10) to identify noninvasive biomarkers reflective of disease activity. RESULTS: 688 human proteins were quantified, with 523 measured in both cohorts. In UC stool 96 proteins were differentially abundant and in CD stool 126 proteins were differentially abundant compared to healthy stool (absolute log2 fold change > 1, p-value < 0.05). Many of these fecal proteins are associated with infiltrating immune cells and ulceration/rectal bleeding, which are hallmarks of IBD pathobiology. Mapping the identified fecal proteins to a whole blood single-cell RNA sequencing data set revealed the involvement of various immune cell subsets to the IBD fecal proteome. CONCLUSIONS AND CLINICAL RELEVANCE: Findings from this study not only confirmed the presence of established fecal biomarkers for IBD, such as calprotectin and lactoferrin, but also revealed new fecal proteins from multiple pathways known to be dysregulated in IBD. These novel proteins could serve as potential noninvasive biomarkers to monitor specific aspects of IBD disease activity which could expedite clinical development of novel therapeutic targets.

2022 White Paper on Recent Issues in Bioanalysis: ICH M10 BMV Guideline & Global Harmonization; Hybrid Assays; Oligonucleotides & ADC; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A - Recommendations on Mass Spectrometry, Chromatography and Sample Preparation, Novel Technologies, Novel Modalities, and Novel Challenges, ICH M10 BMV Guideline & Global Harmonization Part 1B - Regulatory Agencies' Inputs on Regulated Bioanalysis/BMV, Biomarkers/CDx/BAV, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on the ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Mass Spectrometry and ICH M10. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (LBA, Biomarkers/CDx and Cytometry) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 15 and 14 (2023), respectively.

Using SILAC to Develop Quantitative Data-Independent Acquisition (DIA) Proteomic Methods.

Proteins are integral to biological systems and functions. Identifying and quantifying proteins can therefore offer systems-wide insights into protein-protein interactions, cellular signaling, and biological pathway activity. The use of quantitative proteomics has become a method of choice for identifying and quantifying proteins in complex matrices. Proteomics allows researchers to survey hundreds to thousands of proteins in a less biased manner than classical antibody-based protein capture strategies. Typically, discovery approaches have used data-dependent acquisition (DDA) methods, but this approach suffers from stochasticity that compromises quantitation. Recent developments in data-independent acquisition (DIA) proteomics workflows enable proteomic profiling of thousands of samples with increased peak picking consistency making it an excellent candidate for discovering and assessing biomarkers in clinical samples. However, quantitation of peptides from DIA datasets is computationally intensive, and guidelines on how to establish DIA methods are lacking. Method development and optimization require novel tools to visualize and filter DIA datasets appropriately. Here, a protocol and novel script workflow for the optimization of quantitative DIA methods using stable isotope labeling of amino acids in culture (SILAC) are presented. This protocol includes steps for cell growth and labeling, peptide digestion and preparation, and optimization of quantitative DIA methods. In addition, important steps for (1) computational analysis to identify and quantify peptides, (2) data visualizations to identify the linear abundance ranges for all peptides in the sample, and (3) descriptions of how to find high confidence quantitation abundance thresholds are described herein.

Proteomic Discovery of Stool Protein Biomarkers for Distinguishing Pediatric Inflammatory Bowel Disease Flares.

Fecal calprotectin, the heterodimer of S100A8/S100A9, makes up ∼60% of neutrophil cytoplasmic protein content and is a canonical clinical biomarker of gut inflammation.1,2 Fecal calprotectin is commonly used to diagnose inflammatory bowel disease (IBD) and has an average sensitivity of 93% and specificity of 96%.1 For diagnosing pediatric IBD, calprotectin fecal tests have similar sensitivity (92%) but substantially less specificity (76%).1 Low specificity, especially in pediatric patients, means a high likelihood of false negative diagnoses, suggesting that a substantial number of active IBD cases remain undiagnosed and untreated. To improve on calprotectin as an effective IBD monitoring tool in pediatric populations, specificity must be increased. To develop an assay with higher specificity in pediatric patients, we identified a panel of multiple proteins that are present in both IBD flare and remission but have distinct abundance ranges between each condition. From a pilot cohort of 10 pediatric patients in states of either active disease or remission, we discovered and relatively quantified hundreds of stool proteins. Of these, our mass spectrometry-based study prioritized at least 5 that have potential to augment negative predictive power of mucosal-level inflammation alongside fecal biomarkers such as calprotectin.

Latent-period stool proteomic assay of multiple sclerosis model indicates protective capacity of host-expressed protease inhibitors.

Diseases are often diagnosed once overt symptoms arise, ignoring the prior latent period when effective prevention may be possible. Experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, exhibits such disease latency, but the molecular processes underlying this asymptomatic period remain poorly characterized. Gut microbes also influence EAE severity, yet their impact on the latent period remains unknown. Here, we show the latent period between immunization and EAE's overt symptom onset is characterized by distinct host responses as measured by stool proteomics. In particular, we found a transient increase in protease inhibitors that inversely correlated with disease severity. Vancomycin administration attenuated both EAE symptoms and protease inhibitor induction potentially by decreasing immune system reactivity, supporting a subset of the microbiota's role in modulating the host's latent period response. These results strengthen previous evidence of proteases and their inhibitors in EAE and highlight the utility stool-omics for revealing complex, dynamic biology.

Transient Osmotic Perturbation Causes Long-Term Alteration to the Gut Microbiota.

Osmotic diarrhea is a prevalent condition in humans caused by food intolerance, malabsorption, and widespread laxative use. Here, we assess the resilience of the gut ecosystem to osmotic perturbation at multiple length and timescales using mice as model hosts. Osmotic stress caused reproducible extinction of highly abundant taxa and expansion of less prevalent members in human and mouse microbiotas. Quantitative imaging revealed decimation of the mucus barrier during osmotic perturbation, followed by recovery. The immune system exhibited temporary changes in cytokine levels and a lasting IgG response against commensal bacteria. Increased osmolality prevented growth of commensal strains in vitro, revealing one mechanism contributing to extinction. Environmental availability of microbiota members mitigated extinction events, demonstrating how species reintroduction can affect community resilience. Our findings (1) demonstrate that even mild osmotic diarrhea can cause lasting changes to the microbiota and host and (2) lay the foundation for interventions that increase system-wide resilience.

Structure, biochemistry, and inhibition of essential 4'-phosphopantetheinyl transferases from two species of Mycobacteria.

4'-Phosphopantetheinyl transferases (PPTase) post-translationally modify carrier proteins with a phosphopantetheine moiety, an essential reaction in all three domains of life. In the bacterial genus Mycobacteria, the Sfp-type PPTase activates pathways necessary for the biosynthesis of cell wall components and small molecule virulence factors. We solved the X-ray crystal structures and biochemically characterized the Sfp-type PPTases from two of the most prevalent Mycobacterial pathogens, PptT of M. tuberculosis and MuPPT of M. ulcerans. Structural analyses reveal significant differences in cofactor binding and active site composition when compared to previously characterized Sfp-type PPTases. Functional analyses including the efficacy of Sfp-type PPTase-specific inhibitors also suggest that the Mycobacterial Sfp-type PPTases can serve as therapeutic targets against Mycobacterial infections.