3.2 Sample
Collection
1. Fill bead-beating tubes with ~300 mL of zirconia/silica beads
and 1 mL of cold metabolite extraction buffer.
2. Powder dry ice and place in a flat container for sample
collection.
3. Place the bead-beating tubes in a CoolRack on top of the dry
ice to chill the extraction buffer.
4. Using sterile tweezers and a pipette tip, fold bacteria-laden
filters in half twice and submerge in the cold extraction buffer,
one filter per tube (see Note 8). Insure to recap the tubes
quickly so that solvents do not evaporate.
5. Invert each tube a few times to dislodge the bacterial lawn from
the filter.
6. Use tweezers to remove filters from the tubes and discard
them, leaving the bacteria in the extraction buffer along with
the beads.
7. At this stage, samples can be stored in a À80
C freezer for a
few weeks if required.
3.3 Metabolite
Extraction
1. Thaw samples from Subheading 3.2 on ice and place in cooled
bead beater (4
C or colder).
2. Bead beat at 6000 rpm, 6 times, for 30 s each round (see Note
9).
3. Spin samples at 13,000 Â g for 10 min at 4
C to pellet debris.
4. Transfer supernatant to Spin-X tubes and centrifuge at
5000 Â g for 5 min at 4
C.
Transfer the filtrate to sterilized microcentrifuge tubes (see
Note 10).
3.4 Preparation of
Samples for LC-MS
1. Mix 75 μL of sample extracts from Subheading 3.3 with 75 μL
of the acetonitrile–formic acid solution.
2. Vortex to mix and spin at 13,000 Â g for 4 min.
3. Pipette 100 mL supernatant into LC vial and cap.
4. Tap tubes to insure no bubbles are trapped in sample.
5. Load into LC-MS autosampler tray.
6. Proceed with either the standard reversed-phase LC-MS separation method (Subheading 3.5) or ion-pairing LC-MS separation method (Subheading 3.6) (see Note 11).
3.5 Standard
Reversed-Phase LCMS Run
1. Separate samples from Subheading 3.4 on a Cogent Diamond
Hydride Type C column linked to an Agilent Accurate Mass
6220 Time-of-Flight (TOF) spectrometer and coupled with an
Agilent 1200 LC system (or equivalent) (see Note 12).
586
Kyle A. Planck and Kyu Rhee
Collection
1. Fill bead-beating tubes with ~300 mL of zirconia/silica beads
and 1 mL of cold metabolite extraction buffer.
2. Powder dry ice and place in a flat container for sample
collection.
3. Place the bead-beating tubes in a CoolRack on top of the dry
ice to chill the extraction buffer.
4. Using sterile tweezers and a pipette tip, fold bacteria-laden
filters in half twice and submerge in the cold extraction buffer,
one filter per tube (see Note 8). Insure to recap the tubes
quickly so that solvents do not evaporate.
5. Invert each tube a few times to dislodge the bacterial lawn from
the filter.
6. Use tweezers to remove filters from the tubes and discard
them, leaving the bacteria in the extraction buffer along with
the beads.
7. At this stage, samples can be stored in a À80
C freezer for a
few weeks if required.
3.3 Metabolite
Extraction
1. Thaw samples from Subheading 3.2 on ice and place in cooled
bead beater (4
C or colder).
2. Bead beat at 6000 rpm, 6 times, for 30 s each round (see Note
9).
3. Spin samples at 13,000 Â g for 10 min at 4
C to pellet debris.
4. Transfer supernatant to Spin-X tubes and centrifuge at
5000 Â g for 5 min at 4
C.
Transfer the filtrate to sterilized microcentrifuge tubes (see
Note 10).
3.4 Preparation of
Samples for LC-MS
1. Mix 75 μL of sample extracts from Subheading 3.3 with 75 μL
of the acetonitrile–formic acid solution.
2. Vortex to mix and spin at 13,000 Â g for 4 min.
3. Pipette 100 mL supernatant into LC vial and cap.
4. Tap tubes to insure no bubbles are trapped in sample.
5. Load into LC-MS autosampler tray.
6. Proceed with either the standard reversed-phase LC-MS separation method (Subheading 3.5) or ion-pairing LC-MS separation method (Subheading 3.6) (see Note 11).
3.5 Standard
Reversed-Phase LCMS Run
1. Separate samples from Subheading 3.4 on a Cogent Diamond
Hydride Type C column linked to an Agilent Accurate Mass
6220 Time-of-Flight (TOF) spectrometer and coupled with an
Agilent 1200 LC system (or equivalent) (see Note 12).
586
Kyle A. Planck and Kyu Rhee