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

ABSTRACT Stool-based proteomics is capable of significantly augmenting our understanding of host-gut microbe interactions. However, compared to competing technologies, such as metagenomics and 16S rRNA sequencing, it is underutilized due to its low throughput and the negative impact sample contamina...

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Autores principales:	Carlos G. Gonzalez, Hannah C. Wastyk, Madeline Topf, Christopher D. Gardner, Justin L. Sonnenburg, Joshua E. Elias
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	diet fermented fiber high-throughput mass spectrometry metaproteomics Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a1129286bbcc4160b7450732b5bb03bb
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spelling	oai:doaj.org-article:a1129286bbcc4160b7450732b5bb03bb2021-12-02T19:46:20ZHigh-Throughput Stool Metaproteomics: Method and Application to Human Specimens10.1128/mSystems.00200-202379-5077https://doaj.org/article/a1129286bbcc4160b7450732b5bb03bb2020-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00200-20https://doaj.org/toc/2379-5077ABSTRACT Stool-based proteomics is capable of significantly augmenting our understanding of host-gut microbe interactions. However, compared to competing technologies, such as metagenomics and 16S rRNA sequencing, it is underutilized due to its low throughput and the negative impact sample contaminants can have on highly sensitive mass spectrometry equipment. Here, we present a new stool proteomic processing pipeline that addresses these shortcomings in a highly reproducible and quantitative manner. Using this method, 290 samples from a dietary intervention study were processed in approximately 1.5 weeks, largely done by a single researcher. These data indicated a subtle but distinct monotonic increase in the number of significantly altered proteins between study participants on fiber- or fermented food-enriched diets. Lastly, we were able to classify study participants based on their diet-altered proteomic profiles and demonstrated that classification accuracies of up to 89% could be achieved by increasing the number of subjects considered. Taken together, this study represents the first high-throughput proteomic method for processing stool samples in a technically reproducible manner and has the potential to elevate stool-based proteomics as an essential tool for profiling host-gut microbiome interactions in a clinical setting. IMPORTANCE Widely available technologies based on DNA sequencing have been used to describe the kinds of microbes that might correlate with health and disease. However, mechanistic insights might be best achieved through careful study of the dynamic proteins at the interface between the foods we eat, our microbes, and ourselves. Mass spectrometry-based proteomics has the potential to revolutionize our understanding of this complex system, but its application to clinical studies has been hampered by low-throughput and laborious experimentation pipelines. In response, we developed SHT-Pro, the first high-throughput pipeline designed to rapidly handle large stool sample sets. With it, a single researcher can process over one hundred stool samples per week for mass spectrometry analysis, conservatively approximately 10× to 100× faster than previous methods, depending on whether isobaric labeling is used or not. Since SHT-Pro is fairly simple to implement using commercially available reagents, it should be easily adaptable to large-scale clinical studies.Carlos G. GonzalezHannah C. WastykMadeline TopfChristopher D. GardnerJustin L. SonnenburgJoshua E. EliasAmerican Society for Microbiologyarticledietfermentedfiberhigh-throughputmass spectrometrymetaproteomicsMicrobiologyQR1-502ENmSystems, Vol 5, Iss 3 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	diet fermented fiber high-throughput mass spectrometry metaproteomics Microbiology QR1-502
spellingShingle	diet fermented fiber high-throughput mass spectrometry metaproteomics Microbiology QR1-502 Carlos G. Gonzalez Hannah C. Wastyk Madeline Topf Christopher D. Gardner Justin L. Sonnenburg Joshua E. Elias High-Throughput Stool Metaproteomics: Method and Application to Human Specimens
description	ABSTRACT Stool-based proteomics is capable of significantly augmenting our understanding of host-gut microbe interactions. However, compared to competing technologies, such as metagenomics and 16S rRNA sequencing, it is underutilized due to its low throughput and the negative impact sample contaminants can have on highly sensitive mass spectrometry equipment. Here, we present a new stool proteomic processing pipeline that addresses these shortcomings in a highly reproducible and quantitative manner. Using this method, 290 samples from a dietary intervention study were processed in approximately 1.5 weeks, largely done by a single researcher. These data indicated a subtle but distinct monotonic increase in the number of significantly altered proteins between study participants on fiber- or fermented food-enriched diets. Lastly, we were able to classify study participants based on their diet-altered proteomic profiles and demonstrated that classification accuracies of up to 89% could be achieved by increasing the number of subjects considered. Taken together, this study represents the first high-throughput proteomic method for processing stool samples in a technically reproducible manner and has the potential to elevate stool-based proteomics as an essential tool for profiling host-gut microbiome interactions in a clinical setting. IMPORTANCE Widely available technologies based on DNA sequencing have been used to describe the kinds of microbes that might correlate with health and disease. However, mechanistic insights might be best achieved through careful study of the dynamic proteins at the interface between the foods we eat, our microbes, and ourselves. Mass spectrometry-based proteomics has the potential to revolutionize our understanding of this complex system, but its application to clinical studies has been hampered by low-throughput and laborious experimentation pipelines. In response, we developed SHT-Pro, the first high-throughput pipeline designed to rapidly handle large stool sample sets. With it, a single researcher can process over one hundred stool samples per week for mass spectrometry analysis, conservatively approximately 10× to 100× faster than previous methods, depending on whether isobaric labeling is used or not. Since SHT-Pro is fairly simple to implement using commercially available reagents, it should be easily adaptable to large-scale clinical studies.
format	article
author	Carlos G. Gonzalez Hannah C. Wastyk Madeline Topf Christopher D. Gardner Justin L. Sonnenburg Joshua E. Elias
author_facet	Carlos G. Gonzalez Hannah C. Wastyk Madeline Topf Christopher D. Gardner Justin L. Sonnenburg Joshua E. Elias
author_sort	Carlos G. Gonzalez
title	High-Throughput Stool Metaproteomics: Method and Application to Human Specimens
title_short	High-Throughput Stool Metaproteomics: Method and Application to Human Specimens
title_full	High-Throughput Stool Metaproteomics: Method and Application to Human Specimens
title_fullStr	High-Throughput Stool Metaproteomics: Method and Application to Human Specimens
title_full_unstemmed	High-Throughput Stool Metaproteomics: Method and Application to Human Specimens
title_sort	high-throughput stool metaproteomics: method and application to human specimens
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/a1129286bbcc4160b7450732b5bb03bb
work_keys_str_mv	AT carlosggonzalez highthroughputstoolmetaproteomicsmethodandapplicationtohumanspecimens AT hannahcwastyk highthroughputstoolmetaproteomicsmethodandapplicationtohumanspecimens AT madelinetopf highthroughputstoolmetaproteomicsmethodandapplicationtohumanspecimens AT christopherdgardner highthroughputstoolmetaproteomicsmethodandapplicationtohumanspecimens AT justinlsonnenburg highthroughputstoolmetaproteomicsmethodandapplicationtohumanspecimens AT joshuaeelias highthroughputstoolmetaproteomicsmethodandapplicationtohumanspecimens
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High-Throughput Stool Metaproteomics: Method and Application to Human Specimens

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