In this chapter, we describe in detail our metaproteomics workflow for the sample collection, preparation, and mass spectrometry
analysis of intestinal mucus samples. Mucus samples are collected by
gently scraping over the epithelial surface preserving both the host
and microbial component. This method of sample collection is
applicable on resected mouse intestine as well as on patient-derived
biopsies collected during colonoscopy. The minimal sample
handling and use of high-resolution mass spectrometry with a
combined de novo and targeted peptide identification strategy
allow for an unbiased assessment of both host and microbial
proteome.
2 Materials
Prepare all solutions using analytical-grade reagents and liquid
chromatography–mass spectrometry (LC-MS) grade water, acetonitrile (ACN), and methanol (MetOH). Buffers can be stored at
room temperature and used for up to 1 month unless otherwise
stated.
2.1 Collection
of Intestinal Mucus
1. Mucus collection buffer: 2% SDS, 0.1 M Tris–HCl, pH 7.5.
2. Dissection tools: Scissors, forceps, and needles.
3. Silicone-covered plate: Petri dish covered with SYLGARD
®
184 prepared according to the manufacture’s instructions.
4. Low-protein-binding Eppendorf tubes.
5. Isoflurane.
2.2 Sample Lysis
1. 1 M DTT.
2. Ultrasonic probe homogenizer
3. Heating block
4. Benchtop centrifuge.
2.3 Filter-Aided
Sample Preparation
(FASP)
1. Urea buffer A (UA): 8 M urea in 0.1 M Tris–HCl (pH 8.5).
Prepare 1 ml per sample (see Note 1).
2. Urea buffer B (UB): 8 M urea in 0.1 M Tris–HCl (pH 8.0).
Prepare 1 ml per sample (see Note 1).
3. Iodoactetamide (0.05 M IAA) solution in UA. Prepare 0.1 ml
per sample (see Note 2).
4. Endoproteinase Lys-C 0.1μg/μl stock solution in 0.05 M Tris–
HCl (pH 8.5).
5. Sequencing-grade modified trypsin 0.1μg/μl stock solution in
0.05 M acetic acid.
Metaproteomics Analysis of Host–Microbiota Interfaces
169
analysis of intestinal mucus samples. Mucus samples are collected by
gently scraping over the epithelial surface preserving both the host
and microbial component. This method of sample collection is
applicable on resected mouse intestine as well as on patient-derived
biopsies collected during colonoscopy. The minimal sample
handling and use of high-resolution mass spectrometry with a
combined de novo and targeted peptide identification strategy
allow for an unbiased assessment of both host and microbial
proteome.
2 Materials
Prepare all solutions using analytical-grade reagents and liquid
chromatography–mass spectrometry (LC-MS) grade water, acetonitrile (ACN), and methanol (MetOH). Buffers can be stored at
room temperature and used for up to 1 month unless otherwise
stated.
2.1 Collection
of Intestinal Mucus
1. Mucus collection buffer: 2% SDS, 0.1 M Tris–HCl, pH 7.5.
2. Dissection tools: Scissors, forceps, and needles.
3. Silicone-covered plate: Petri dish covered with SYLGARD
®
184 prepared according to the manufacture’s instructions.
4. Low-protein-binding Eppendorf tubes.
5. Isoflurane.
2.2 Sample Lysis
1. 1 M DTT.
2. Ultrasonic probe homogenizer
3. Heating block
4. Benchtop centrifuge.
2.3 Filter-Aided
Sample Preparation
(FASP)
1. Urea buffer A (UA): 8 M urea in 0.1 M Tris–HCl (pH 8.5).
Prepare 1 ml per sample (see Note 1).
2. Urea buffer B (UB): 8 M urea in 0.1 M Tris–HCl (pH 8.0).
Prepare 1 ml per sample (see Note 1).
3. Iodoactetamide (0.05 M IAA) solution in UA. Prepare 0.1 ml
per sample (see Note 2).
4. Endoproteinase Lys-C 0.1μg/μl stock solution in 0.05 M Tris–
HCl (pH 8.5).
5. Sequencing-grade modified trypsin 0.1μg/μl stock solution in
0.05 M acetic acid.
Metaproteomics Analysis of Host–Microbiota Interfaces
169