3D HPLC-MS with Reversed-Phase Separation Functionality in All Three Dimensions for Large-Scale Bottom-Up Proteomics and Peptide Retention Data Collection

The growing complexity of proteomics samples and the desire for deeper analysis drive the development of both better MS instrument and advanced multidimensional separation schemes. We applied 1D, 2D, and 3D LC-MS/MS separation protocols (all of reversed-phase C18 functionality) to a tryptic digest of whole Jurkat cell lysate to estimate the depth of proteome coverage and to collect high-quality peptide retention information. We varied pH of the eluent and hydrophobicity of ion-pairing modifier to achieve good separation orthogonality (utilization of MS instrument time). All separation modes employed identical LC settings with formic-acid-based eluents in the last dimension. The 2D protocol used a high pH–low pH scheme with 21 concatenated fractions. In the 3D protocol, six concatenated fractions from the first dimension (C18, heptafluorobutyric acid) were analyzed using the identical 2D LC-MS procedure. This approach permitted a detailed evaluation of the analysis output consuming 21× and 126× the analysis time and sample load compared to 1D. Acquisition over 189 h of instrument time in 3D mode resulted in the identification of ∼14 000 proteins and ∼250 000 unique peptides. We estimated the dynamic range via peak intensity at the MS² level as approximately 10^4.2, 10^5.6, and 10^6.2 for the 1D, 2D, and 3D protocols, respectively. The uniform distribution of the number of acquired MS/MS, protein, and peptide identifications across all 126 fractions and through the chromatographic time scale in the last LC-MS stage indicates good separation orthogonality. The protocol is scalable and is amenable to the use of peptide retention prediction in all dimensions. All these features make it a very good candidate for large-scale bottom-up proteomic runs, which target both protein identification as well as the collection of peptide retention data sets for targeted quantitative applications

OP29 Peri-natal exposure to parental Crohn’s disease is associated with impaired gut barrier, microbiome composition differences and increased risk of Crohn’s disease

Abstract Background Previous studies have shown that offspring of Crohn’s disease (CD) patients have a 6-8 fold risk of incident CD than the general population. Also, newborns of women with CD (vs healthy women) have altered stool microbiome composition and elevated fecal calprotectin (FCP). The "peri-natal period" (pregnancy and first year postpartum) is critical for the development and maturation of the gut microbiome as well as immune responses. However, it is unclear if exposure to parental CD diagnosis during the peri-natal period is associated with increased risk of CD. We aimed to investigate whether peri-natal exposure to parental CD (compared to exposure later in life) has an impact on offspring’s gut barrier function, microbiome composition, and the offspring’s risk of incident CD. Methods We assessed 1252 healthy offspring (6-35 years of age) of patients with CD who were recruited in the CCC-GEM Project, a large prospective cohort study following healthy FDRs of CD patients. We classified offspring into "peri-natally exposed" or "exposed later in life" to parental CD diagnosis. We measured baseline FCP and urinary fractional excretion ratio of lactulose to mannitol (marker of intestinal permeability, LMR). Fecal microbiome composition was determined by 16S rRNA gene sequencing. General estimating equation regression and Cox-proportional hazard modeling was used to assess if peri-natal exposure to parental CD is associated with LMR, FCP, altered microbial composition, and future CD onset. Offspring’s age and sex and family clusters were accounted for in the models. Results Offspring exposed to a CD parent peri-natally have a 4.73 fold higher risk (aHR 95%CI 1.28-17.46) of developing CD compared to those exposed to a parent who developed CD later in life (11/586 vs 3/666). Baseline LMR was significantly higher in the offspring exposed to parental CD peri-natally (p=0.003) while no significant difference was observed for FCP at recruitment (p=0.94). We identified five bacterial taxa that were significantly increased in the peri-natally exposed group (FDR-adjusted p<0.05). In a subgroup of offspring of mothers with CD, a dose effect between exposure age and risk was seen; the earlier the offspring was exposed to mother’s CD diagnosis peri-natally, the higher the offspring’s risk of CD development; this trend was not seen in offspring of fathers with CD. Conclusion Offspring exposed to parental CD peri-natally had a higher risk of developing CD than those exposed to parental CD later in life. Early life exposure to parental CD was associated with higher baseline LMR and altered bacterial taxa. These results suggest that environmental exposure during the critical peri-natal period may determine the offspring’s future risk of developing CD

Mediterranean-Like Dietary Pattern Associations With Gut Microbiome Composition and Subclinical Gastrointestinal Inflammation

BACKGROUND & AIMS: Case-control studies have shown that patients with Crohn's disease (CD) have a microbial composition different from healthy individuals. Although the causes of CD are unknown, epidemiologic studies suggest that diet is an important contributor to CD risk, potentially via modulation of bacterial composition and gut inflammation. We hypothesized that long-term dietary clusters (DCs) are associated with gut microbiome compositions and gut inflammation. Our objectives were to identify dietary patterns and assess whether they are associated with alterations in specific gut microbial compositions and subclinical levels of gut inflammation in a cohort of healthy first-degree relatives (FDRs) of patients with CD. METHODS: As part of the Genetic, Environmental, Microbial (GEM) Project, we recruited a cohort of 2289 healthy FDRs of patients with CD. Individuals provided stool samples and answered a validated food frequency questionnaire reflecting their habitual diet during the year before sample collection. Unsupervised analysis identified 3 dietary and 3 microbial composition clusters. RESULTS: DC3, resembling the Mediterranean diet, was strongly associated with a defined microbial composition, with an increased abundance of fiber-degrading bacteria, such as Ruminococcus, as well as taxa such as Faecalibacterium. The DC3 diet was also significantly associated with lower levels of subclinical gut inflammation, defined by fecal calprotectin, compared with other dietary patterns. No significant associations were found between individual food items and fecal calprotectin, suggesting that long-term dietary patterns rather than individual food items contribute to subclinical gut inflammation. Additionally, mediation analysis demonstrated that DC3 had a direct effect on subclinical inflammation that was partially mediated by the microbiota. CONCLUSIONS: Overall, these results indicated that Mediterranean-like dietary patterns are associated with microbiome and lower intestinal inflammation. This study will help guide future dietary strategies that affect microbial composition and host gut inflammation to prevent diseases

Healthy first-degree relatives from multiplex families versus simplex families have a higher subclinical intestinal inflammation, a distinct fecal microbial signature, and harbor a higher risk of developing Crohn’s disease

Unaffected first-degree relatives (FDRs) from families with two or more affected FDRs with Crohn’s disease (CD, multiplex families) have a high risk of developing CD, although the underlying mechanisms driving this risk are poorly understood. We aimed to identify differences in biomarkers between FDRs from multiplex versus simplex families and to investigate the risk of future CD onset accounting for potential confounders. We assessed the Crohn’s and Colitis Canada Genetic Environmental Microbial (CCC-GEM) cohort of healthy FDRs of patients with CD. Genome-wide CD-polygenic risk scores (CD-PRS), urinary fractional excretion of lactulose-to-mannitol ratio (LMR), fecal calprotectin (FCP), and fecal 16S ribosomal RNA microbiome were measured at recruitment. Associations between CD multiplex status and baseline biomarkers were determined using generalized estimating equations models. Cox models were used to assess the risk of future CD onset. There were 4051 participants from simplex families and 334 from CD multiplex families. CD multiplex status was significantly associated with higher baseline FCP (p=0.026) but not with baseline CD-PRS or LMR. Three bacterial genera were found to be differentially abundant between both groups. CD multiplex status at recruitment was independently associated with an increased risk of developing CD (adjusted hazard ratio 3.65, 95% confidence interval 2.18 – 6.11, p < 0.001). Within FDRs of patients with CD, participants from multiplex families had a 3-fold increased risk of CD onset, a higher FCP, and an altered bacterial composition, but not genetic burden or altered gut permeability. These results suggest that putative environmental factors might be enriched in FDRs from multiplex families

Environmental Factors Associated with Risk of Crohn’s Disease Development in the CCC-GEM Project

To date, it is unclear how environmental factors influence Crohn’s Disease (CD) risk and how they interact with biological processes. This study investigates the association between environmental exposures and CD risk and evaluates their association with pre-disease biomarkers. We studied 4,289 healthy first-degree relatives (FDRs) of CD patients from the Crohn's and Colitis Canada - Genetic, Environmental, Microbial (CCC-GEM) project. Regression models identified environmental factors associated with future CD onset and their association with pre-disease biological factors, including altered intestinal permeability measured by urinary fractional excretion of lactulose to mannitol ratio(LMR); gut inflammation via fecal calprotectin(FCP) levels; and fecal microbiome composition through 16S rRNA sequencing. Over a 5.62-year median follow-up, 86 FDRs developed CD. Living with a dog between ages 5-15 (HR=0.62; 95% CI=0.40-0.96; P = .034), and living with a large family size in the first year of life (HR=0.43; 95% CI=0.21-0.85; P = .016) were associated with decreased CD risk; whereas having a bird at the time of recruitment (HR=2.78; CI=1.36-5.68; P = .005) was associated with an increased CD risk. Furthermore, living with a dog was associated with reduced LMR, altered relative abundance of multiple bacterial genera, and increased Chao1 diversity; while bird owners had higher FCP levels. Large family during participants' first year of life was associated with altered microbiota composition without affecting FCP or LMR. This study identifies environmental variables associated with CD risk. These variables were also associated with altered barrier function, subclinical inflammation, and gut microbiome composition shifts, suggesting potential roles in CD pathogenesis