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...