Chapter 10
Metaproteomics Analysis of Host–Microbiota Interfaces
Sjoerd van der Post and Liisa Arike
Abstract
Metaproteomics of host–microbiome interfaces comprises the analysis of complex mixtures of bacteria,
archaea, fungi, and viruses in combination with its host cells. Microbial niches can be found all over the host
including the skin, oral cavity, and the intestine and are considered to be essential for the homeostasis. The
complex interactions between the host and diverse commensal microbiota are poorly characterized while of
great interest as dysbiosis is associated with the development of various inflammatory and metabolic
diseases. The metaproteomics workflows to study these interfaces are currently being established, and
many challenges remain. The major challenge is the large diversity in species composition that make up the
microbiota, which results in complex samples that require extended mass spectrometry analysis time. In
addition, current database search strategies are not developed to the size of the search space required for
unbiased microbial protein identification.
Here, we describe a workflow for the proteomics analysis of microbial niches with a focus on intestinal
mucus layer. We will cover step-by-step the sample collection, sample preparation, liquid chromatography–mass spectrometry, and data analysis.
Key words Microbiota, Proteomics, Mass spectrometry, Metaproteomics, Intestine, Mucus
1 Introduction
The mucus layer that covers the epithelium along the gastrointestinal tract is the first site of interaction between the host and the
luminal microbiota. Mucus is a complex protein network organized
around the MUC2 mucin acting as the first line of defense against
bacterial invasion but also providing a habitat for the microbiota
[1]. The commensal microbiota supports the hosts with nutrients
and vitamins, primes the immune system, and prevents invasion by
pathogens. An imbalance in gut microbiota has been associated
with inflammatory bowel disease, type 1 diabetes, and nonalcoholic
fatty liver disease [2]. Despite the progress made by metagenomic
research describing the composition of the gut microbiota, the
current understanding is that the functional capacity rather than
solely the microbial composition is what actually is determinant for
intestinal homeostasis. For that reason, integration of data from
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
167
Metaproteomics Analysis of Host–Microbiota Interfaces
Sjoerd van der Post and Liisa Arike
Abstract
Metaproteomics of host–microbiome interfaces comprises the analysis of complex mixtures of bacteria,
archaea, fungi, and viruses in combination with its host cells. Microbial niches can be found all over the host
including the skin, oral cavity, and the intestine and are considered to be essential for the homeostasis. The
complex interactions between the host and diverse commensal microbiota are poorly characterized while of
great interest as dysbiosis is associated with the development of various inflammatory and metabolic
diseases. The metaproteomics workflows to study these interfaces are currently being established, and
many challenges remain. The major challenge is the large diversity in species composition that make up the
microbiota, which results in complex samples that require extended mass spectrometry analysis time. In
addition, current database search strategies are not developed to the size of the search space required for
unbiased microbial protein identification.
Here, we describe a workflow for the proteomics analysis of microbial niches with a focus on intestinal
mucus layer. We will cover step-by-step the sample collection, sample preparation, liquid chromatography–mass spectrometry, and data analysis.
Key words Microbiota, Proteomics, Mass spectrometry, Metaproteomics, Intestine, Mucus
1 Introduction
The mucus layer that covers the epithelium along the gastrointestinal tract is the first site of interaction between the host and the
luminal microbiota. Mucus is a complex protein network organized
around the MUC2 mucin acting as the first line of defense against
bacterial invasion but also providing a habitat for the microbiota
[1]. The commensal microbiota supports the hosts with nutrients
and vitamins, primes the immune system, and prevents invasion by
pathogens. An imbalance in gut microbiota has been associated
with inflammatory bowel disease, type 1 diabetes, and nonalcoholic
fatty liver disease [2]. Despite the progress made by metagenomic
research describing the composition of the gut microbiota, the
current understanding is that the functional capacity rather than
solely the microbial composition is what actually is determinant for
intestinal homeostasis. For that reason, integration of data from
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
167