6. Ammonium bicarbonate (ABC) 0.05 M solution in water.
Prepare 0.25 ml per sample.
7. Nanosep 30K Omega centrifugal devices.
8. Benchtop centrifuge.
9. Thermomixer.
10. Microvolume spectrophotometer.
2.4 C18-StageTip
Cleanup
1. Buffer A: 0.1% trifloroacetic acid (TFA) in water.
2. Buffer B: 80% ACN, 0.1% TFA in water.
3. C18-StageTips (see Note 3).
4. 2-ml Eppendorf tubes.
5. Vacuum concentrator.
2.5 LC-MS
1. Buffer A: 0.1% formic acid (FA) in water.
2. Buffer B: 80% ACN, 0.1% FA in water.
3. EASY-Spray C18 column (50 cm long, 75μm inner diameter,
2μm particle size) or equivalent reversed-phase nano column.
4. EASY-nLC 1200 or alternative nano LC system.
5. Q-Exactive HF-X or other tandem mass spectrometer.
2.6 Computational
Resources
and Required Software
1. Windows OS, 8 core CPU, 32 GB of RAM, and 1 TB SSD for
data processing and storage.
2. PEAKS Studio v8.5 or alternative de novo peptide search
engine:
http://www.bioinfor.com/peaks-studio/
3. Python v3.6 or higher:
https://www.python.org/downloads/
4. ProteoClade for taxa-specific peptide annotation:
https://github.com/HeldLab/ProteoClade
5. MaxQuant or alternative targeted database search engine:
https://maxquant.org/
6. NotePad++ for file inspection and editing:
https://notepad-plus-plus.org
3 Methods
3.1 Collection of
Mucus Sample
The mucus samples are directly collected after sectioning the colon
to prevent further bacterial growth. The described protocol is
focused on the colon but can be applied to all other regions of
the intestine. Mucus-associated bacteria are retained, by only
removing the fecal pellets without any further flushing of the tissue.
170
Sjoerd van der Post and Liisa Arike
Prepare 0.25 ml per sample.
7. Nanosep 30K Omega centrifugal devices.
8. Benchtop centrifuge.
9. Thermomixer.
10. Microvolume spectrophotometer.
2.4 C18-StageTip
Cleanup
1. Buffer A: 0.1% trifloroacetic acid (TFA) in water.
2. Buffer B: 80% ACN, 0.1% TFA in water.
3. C18-StageTips (see Note 3).
4. 2-ml Eppendorf tubes.
5. Vacuum concentrator.
2.5 LC-MS
1. Buffer A: 0.1% formic acid (FA) in water.
2. Buffer B: 80% ACN, 0.1% FA in water.
3. EASY-Spray C18 column (50 cm long, 75μm inner diameter,
2μm particle size) or equivalent reversed-phase nano column.
4. EASY-nLC 1200 or alternative nano LC system.
5. Q-Exactive HF-X or other tandem mass spectrometer.
2.6 Computational
Resources
and Required Software
1. Windows OS, 8 core CPU, 32 GB of RAM, and 1 TB SSD for
data processing and storage.
2. PEAKS Studio v8.5 or alternative de novo peptide search
engine:
http://www.bioinfor.com/peaks-studio/
3. Python v3.6 or higher:
https://www.python.org/downloads/
4. ProteoClade for taxa-specific peptide annotation:
https://github.com/HeldLab/ProteoClade
5. MaxQuant or alternative targeted database search engine:
https://maxquant.org/
6. NotePad++ for file inspection and editing:
https://notepad-plus-plus.org
3 Methods
3.1 Collection of
Mucus Sample
The mucus samples are directly collected after sectioning the colon
to prevent further bacterial growth. The described protocol is
focused on the colon but can be applied to all other regions of
the intestine. Mucus-associated bacteria are retained, by only
removing the fecal pellets without any further flushing of the tissue.
170
Sjoerd van der Post and Liisa Arike