4. Mucus is transparent and sticky; therefore, additional buffer is
needed for collecting it from the tissue and transferring it from
the spatula into the collection tube. Do not apply too much
force when scraping, to prevent contamination with epithelial
cells. The mucus collected from the tissue should be transparent and give some resistance when pipetting (Fig. 1b).
5. Trypsin stock solutions are stored in 50 mM acetic acid, which
inhibits the enzymatic activity and requires activation prior to
digestion. Trypsin is activated by raising the pH by diluting the
stock solution with ABC. Heating at 30
C prior to activation
helps to maximize the enzymatic activity.
6. It is important to prevent the disks from drying out during the
centrifugation steps.
7. It is good practice to remove the methanol and acetonitrile at
the bottom of the tube before introducing the sample as contact between the eluate and StageTip can cause unwanted
sample elution due to the organic solvents.
8. Each Empore disk is able to bind approximately 5μg of peptides; when loading 15μg of peptides, three layers have sufficient binding capacity.
9. Samples can be stored on StageTips for an extended time at
À20
C and eluted just before LC-MS analysis.
10. Final volume should be around 5μl as it is not recommended to
dry the sample completely to avoid peptide loss.
11. If samples have completely dried during concentration, solubilization for 5 min in an ultrasonication bath is recommended.
12. Suggested mass spectrometry settings: MS resolution 60,000,
AGC target 3 Â 10
6 , maximum IT 20 ms, scan range
350–1600 m/z. MS/MS resolution 15,000, AGC target
1 Â 10
5 , maximum IT 50 ms, intensity threshold 1 Â 10
5 ,
top 12, dynamic exclusion 30 s.
13. Conversion of mass spectrometry data into peaklist, which in
some cases is required for the submission to peptide search
engines, can be done using msConvert. The software is able to
convert spectral data from various mass spectrometry platforms
and can be obtained via the link below. The fragmentation
spectra data should be processed in centroid mode and recommended to be filtered from noise using a top “n” most intense
peaks setting. http://proteowizard.sourceforge.net/tools.
shtml
14. Alternative open-source de novo search engines to consider are
PepNovo+ (http://proteomics.ucsd.edu/Software/PepNovo/)
and pNovo (http://pfind.ict.ac.cn/software/pNovo/index.
html).
Metaproteomics Analysis of Host–Microbiota Interfaces
177
needed for collecting it from the tissue and transferring it from
the spatula into the collection tube. Do not apply too much
force when scraping, to prevent contamination with epithelial
cells. The mucus collected from the tissue should be transparent and give some resistance when pipetting (Fig. 1b).
5. Trypsin stock solutions are stored in 50 mM acetic acid, which
inhibits the enzymatic activity and requires activation prior to
digestion. Trypsin is activated by raising the pH by diluting the
stock solution with ABC. Heating at 30
C prior to activation
helps to maximize the enzymatic activity.
6. It is important to prevent the disks from drying out during the
centrifugation steps.
7. It is good practice to remove the methanol and acetonitrile at
the bottom of the tube before introducing the sample as contact between the eluate and StageTip can cause unwanted
sample elution due to the organic solvents.
8. Each Empore disk is able to bind approximately 5μg of peptides; when loading 15μg of peptides, three layers have sufficient binding capacity.
9. Samples can be stored on StageTips for an extended time at
À20
C and eluted just before LC-MS analysis.
10. Final volume should be around 5μl as it is not recommended to
dry the sample completely to avoid peptide loss.
11. If samples have completely dried during concentration, solubilization for 5 min in an ultrasonication bath is recommended.
12. Suggested mass spectrometry settings: MS resolution 60,000,
AGC target 3 Â 10
6 , maximum IT 20 ms, scan range
350–1600 m/z. MS/MS resolution 15,000, AGC target
1 Â 10
5 , maximum IT 50 ms, intensity threshold 1 Â 10
5 ,
top 12, dynamic exclusion 30 s.
13. Conversion of mass spectrometry data into peaklist, which in
some cases is required for the submission to peptide search
engines, can be done using msConvert. The software is able to
convert spectral data from various mass spectrometry platforms
and can be obtained via the link below. The fragmentation
spectra data should be processed in centroid mode and recommended to be filtered from noise using a top “n” most intense
peaks setting. http://proteowizard.sourceforge.net/tools.
shtml
14. Alternative open-source de novo search engines to consider are
PepNovo+ (http://proteomics.ucsd.edu/Software/PepNovo/)
and pNovo (http://pfind.ict.ac.cn/software/pNovo/index.
html).
Metaproteomics Analysis of Host–Microbiota Interfaces
177