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

González, Carlos Gutiérrez; Tankou, Stephanie; Cox, Laura M.; Casavant, Ellen P.; Weiner, Howard L.; Elias, Joshua E.

doi:10.1038/s41598-019-48495-5

Cited by 12 publications

(38 citation statements)

References 65 publications

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“…A major limitation to large-scale adoption of stool metaproteomics has been is its heavily reduced throughput compared to DNA-sequencing. Considering the labor-intensive, multi-day nature of our previously published stool proteomics method, we found that one researcher could reasonably process 25 stool samples per week on average 6,10 . To narrow this gap, we developed our pipeline to rapidly process hundreds of stool samples (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Samples were prepared as described in Gonzalez et al 6 . Briefly, sample pellets were disrupted using 500 uL 8M urea lysis buffer supplemented with Roche cOmplete protease inhibitor (04693159001 ROCHE) by vortexing.…”

Section: Methodsmentioning

confidence: 99%

“… A) Generalized experimental pipeline of our previous workflow. A more extensive description of the method is found in Gonzalez et al 6 . B) Experimental outline designed to test SHT-Pro reproducibility.…”

Section: Si Figsmentioning

confidence: 99%

“…One major hindrance to broader implementation has been low sample processing throughput. Indeed, while we and others previously demonstrated the power and utility of stool proteomics, those studies relied on data generated at rates as low as 10-30 samples per week 6–9 . Additionally, workflows developed for processing for cell culture and tissue lysates are not optimized to eliminate contaminating molecules that are abundant in stool.…”

Section: Introductionmentioning

confidence: 99%

“…As a first demonstration of this method, we applied SHT-Pro to 145 stool specimens longitudinally collected from 29 human participants as part of an ongoing dietary intervention study investigating the biological effects of diets enriched in fiber versus fermented foods (ClinicalTrials.gov Identifier: NCT03275662). Processing them in duplicate (290 total samples) using SHT-Pro took approximately one week from stool to mass-spectrometry-ready peptides, an estimated time savings of over 2.5 months compared to our previously published workflow 5,6 . The resulting data set identified over 5,600 unique host and microbial proteins, 45% which were shared between both study groups.…”

Section: Introductionmentioning

confidence: 99%

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High-Throughput Stool Metaproteomics: Method and Application to Human Specimens

González

Wastyk

Topf

et al. 2020

Preprint

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16Stool-based proteomics is capable of significantly augmenting our understanding of 17 host-gut microbe interactions. However, in comparison to competing technologies such 18 as metagenomics and 16S rRNA sequencing, it is under-utilized due to its low 19 throughput and the negative impact sample contaminants can have on highly sensitive 20 mass spectrometry equipment. Here, we present a new stool proteomic processing 21 pipeline that addresses these shortcomings in a highly reproducible and quantitative 22 manner. Using this method, 290 samples from a dietary intervention study were 23 processed in approximately 1.5 weeks, largely done by a single researcher. These data 24 indicated a subtle but distinct monotonic increase in the number of significantly altered 25 proteins between study participants on fiber-or fermented food-enriched diets. Lastly, 26we were able to classify study participants based on their diet-altered proteomic profiles, 27 and demonstrated that classification accuracies of up to 89% could be achieved by 28 increasing the number of subjects considered. Taken together, this study represents the 29 first high throughout proteomic method for processing stool samples in a technically 30 reproducible manner, and has the potential to elevate stool-based proteomics as an 31 essential tool for profiling host-gut microbiome interactions in a clinical setting. 32 Importance 33Widely available technologies based on DNA sequencing have been used to describe 34 the kinds of microbes that might correlate with health and disease. However, 35 mechanistic insight might be best achieved through careful study of the dynamic 36 proteins at the interface between the foods we eat, our microbes, and ourselves. Mass-37 spectrometry-based proteomics has the potential to revolutionize our understanding of 38 this complex system but its application to clinical studies has been hampered by low-39 throughput and laborious experimentation pipelines. In response, we developed SHT-40Pro, the first high-throughput pipeline designed to rapidly handle large stool sample 41 sets. With it, a single researcher can process over one hundred stool samples per week 42for mass spectrometry analysis, roughly 10 times faster than previous methods. Since 43 SHT-Pro is fairly simple to implement using commercially available reagents, it should 44 be easily adaptable to large-scale clinical studies. 45 human health and disease, and has been associated with disorders ranging from 48 inflammatory bowel disease to autism 1,2 . Stool is a biologically rich biomaterial, 49 containing host, microbe, and dietary proteins, among a rich array of biomolecules. The 50 broad proteinaceous representation of relevant biological entities and interactions, in 51 conjunction with non-invasive sample collection, makes stool ideal for studying the 52 complex ecosystem at the host-gut microbe interface 3 . Microbiome composition can be 53 readily determined using 16S rRNA gene sequencing from stool DNA, and due to its 54 high-throughput nature, is well-suite...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Si Figsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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High-Throughput Stool Metaproteomics: Method and Application to Human Specimens

González

Wastyk

Topf

et al. 2020

Preprint

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Histological and Top-Down Proteomic Analyses of the Visual Pathway in the Cuprizone Demyelination Model

et al. 2022

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A change in visual perception is a frequent early symptom of multiple sclerosis (MS), the pathoaetiology of which remains unclear. Following a slow demyelination process caused by 12 weeks of low-dose (0.1%) cuprizone (CPZ) consumption, histology and proteomics were used to investigate components of the visual pathway in young adult mice. Histological investigation did not identify demyelination or gliosis in the optic tracts, pretectal nuclei, superior colliculi, lateral geniculate nuclei or visual cortices. However, top-down proteomic assessment of the optic nerve/tract revealed a significant change in the abundance of 34 spots in high-resolution two-dimensional (2D) gels. Subsequent liquid chromatography-tandem mass spectrometry (LC-TMS) analysis identified alterations in 75 proteoforms. Literature mining revealed the relevance of these proteoforms in terms of proteins previously implicated in animal models, eye diseases and human MS. Importantly, 24 proteoforms were not previously described in any animal models of MS, eye diseases or MS itself. Bioinformatic analysis indicated involvement of these proteoforms in cytoskeleton organization, metabolic dysregulation, protein aggregation and axonal support. Collectively, these results indicate that continuous CPZ-feeding, which evokes a slow demyelination, results in proteomic changes that precede any clear histological changes in the visual pathway and that these proteoforms may be potential early markers of degenerative demyelinating conditions.

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