4. Add 50μl of buffer A, and centrifuge at 400 Â g for 4 min (see
Note 6).
5. Discard the content of the Eppendorf tube (see Note 7).
6. Add 50μl of buffer A, and centrifuge at 400 Â g for 4 min.
7. Add 15μg of peptides (based on absorbance measurement at
280 nm) in 100–200μl final volume (adjust with buffer A) (see
Note 8).
8. Centrifuge at 400 Â g for 10 min or until the sample has passed
through the filter.
9. Add 50μl of buffer A, and centrifuge at 400 Â g for 4 min.
10. Elute peptides by adding 20μl of buffer B and collecting the
eluate to a new low-protein-binding Eppendorf tube (see
Note 9).
11. Repeat step 10.
12. Dry samples in a vacuum concentrator (see Note 10).
13. Dissolve sample in 40μl of 0.1% FA (see Note 11).
3.5 Peptide Analysis
by Liquid
ChromatographyMass Spectrometry
A recent study evaluated different LC methods for metaproteomics
analysis and concluded that a simple one-column setup in combination with a long column and extended gradient performs similar
to more complex two-dimensional LC setups [14]. Here, we have
used a 3-h gradient and 50-cm column; however, additional sample
fractionation or enrichment strategies could be added to the workflow to increase the depth of analysis. The parameters for LC-MS
analysis are highly dependent on the available platform. The steps
below are therefore only broad recommendations applicable to
most LC-MS systems. Two key features are the use of an extended
LC gradient as the samples are highly complex and adjusting the
ion accumulation time for the fragmentation spectra acquisition to
increase the quality of the de novo peptide identifications and
thereby the overall experimental outcome.
1. Set up and condition the LC column according to the manufacturer’s instructions.
2. Suggested gradient 3–25% B over 175 min, 25–45% B over
30 min, and 45–100% B over 5 min and hold at 100% for
20 min; flow rate 250 nl/min; 40
C column temperature
when column heater is available.
3. Perform mass spectrometry analysis of the samples. Increasing
the ion accumulation time for the collection of fragmentation
spectra is recommended (see Fig. 2a). General settings for Q
Exactive mass spectrometers (Thermo) are provided (see
Note 12).
Metaproteomics Analysis of Host–Microbiota Interfaces
173
Note 6).
5. Discard the content of the Eppendorf tube (see Note 7).
6. Add 50μl of buffer A, and centrifuge at 400 Â g for 4 min.
7. Add 15μg of peptides (based on absorbance measurement at
280 nm) in 100–200μl final volume (adjust with buffer A) (see
Note 8).
8. Centrifuge at 400 Â g for 10 min or until the sample has passed
through the filter.
9. Add 50μl of buffer A, and centrifuge at 400 Â g for 4 min.
10. Elute peptides by adding 20μl of buffer B and collecting the
eluate to a new low-protein-binding Eppendorf tube (see
Note 9).
11. Repeat step 10.
12. Dry samples in a vacuum concentrator (see Note 10).
13. Dissolve sample in 40μl of 0.1% FA (see Note 11).
3.5 Peptide Analysis
by Liquid
ChromatographyMass Spectrometry
A recent study evaluated different LC methods for metaproteomics
analysis and concluded that a simple one-column setup in combination with a long column and extended gradient performs similar
to more complex two-dimensional LC setups [14]. Here, we have
used a 3-h gradient and 50-cm column; however, additional sample
fractionation or enrichment strategies could be added to the workflow to increase the depth of analysis. The parameters for LC-MS
analysis are highly dependent on the available platform. The steps
below are therefore only broad recommendations applicable to
most LC-MS systems. Two key features are the use of an extended
LC gradient as the samples are highly complex and adjusting the
ion accumulation time for the fragmentation spectra acquisition to
increase the quality of the de novo peptide identifications and
thereby the overall experimental outcome.
1. Set up and condition the LC column according to the manufacturer’s instructions.
2. Suggested gradient 3–25% B over 175 min, 25–45% B over
30 min, and 45–100% B over 5 min and hold at 100% for
20 min; flow rate 250 nl/min; 40
C column temperature
when column heater is available.
3. Perform mass spectrometry analysis of the samples. Increasing
the ion accumulation time for the collection of fragmentation
spectra is recommended (see Fig. 2a). General settings for Q
Exactive mass spectrometers (Thermo) are provided (see
Note 12).
Metaproteomics Analysis of Host–Microbiota Interfaces
173