2.6 Acetone–
Methanol Protein
Precipitation
and Extraction
1. Ice-cold acetone/methanol (8:1).
2. Ice-cold 80% acetone.
3. Denaturation buffer (see Note 3).
4. Centrifuge.
5. Bradford reagent.
2.7 Reduction
Dithiothreitol (DTT).
2.8 Alkylation
Iodoacetamide (IAA).
2.9 Predigestion
Lys-C (MS-grade).
2.10 Tryptic
Digestion
1. Trypsin (MS-grade).
2. Formic acid (FA).
3. pH strips.
2.11 Desalting
1. C18 Extraction disks (see Note 4).
2. Speed-vac.
3. 2 mL Glass vials and 250 μL glass inserts.
4. 2% Acetonitrile, 0.1% FA (Solvent A).
5. Methanol (solvent B).
6. 60% Acetonitrile, 0.1% FA (Solvent C).
3 Methods
3.1 Mycobacterial
Strain
Culture M. smegmatis (strain mc
2
155) from frozen stocks in 7H9
media. Upon reaching mid-log phase (OD 600 ¼ 0.8), inoculate
cells on to solid complete 7H10 media. Streak ten times serial
dilutions and leave to grow until single colonies form (about
7–15 days). Pick single colonies using a cell scraper (careful not
to pinch through the agar) and store at À80
until protein
extraction.
3.2 Cell Lysis
1. Suspend single colonies in 1 mL lysis buffer.
2. Disrupt cells by sonication at maximum power for 6 cycles of
30 s with cooling intervals of 1 min on ice, between each cycle.
3. Remove cellular debris by centrifugation at 4000 Â g for
10 min and filter sterilize lysate through 0.2μM filter into a
fresh tube.
4. Store lysates at À20
until needed or proceed with protein
extraction.
Mass Spectrometry-Based Analysis of Mycobacterial Single-Colony Proteome
183
Methanol Protein
Precipitation
and Extraction
1. Ice-cold acetone/methanol (8:1).
2. Ice-cold 80% acetone.
3. Denaturation buffer (see Note 3).
4. Centrifuge.
5. Bradford reagent.
2.7 Reduction
Dithiothreitol (DTT).
2.8 Alkylation
Iodoacetamide (IAA).
2.9 Predigestion
Lys-C (MS-grade).
2.10 Tryptic
Digestion
1. Trypsin (MS-grade).
2. Formic acid (FA).
3. pH strips.
2.11 Desalting
1. C18 Extraction disks (see Note 4).
2. Speed-vac.
3. 2 mL Glass vials and 250 μL glass inserts.
4. 2% Acetonitrile, 0.1% FA (Solvent A).
5. Methanol (solvent B).
6. 60% Acetonitrile, 0.1% FA (Solvent C).
3 Methods
3.1 Mycobacterial
Strain
Culture M. smegmatis (strain mc
2
155) from frozen stocks in 7H9
media. Upon reaching mid-log phase (OD 600 ¼ 0.8), inoculate
cells on to solid complete 7H10 media. Streak ten times serial
dilutions and leave to grow until single colonies form (about
7–15 days). Pick single colonies using a cell scraper (careful not
to pinch through the agar) and store at À80
until protein
extraction.
3.2 Cell Lysis
1. Suspend single colonies in 1 mL lysis buffer.
2. Disrupt cells by sonication at maximum power for 6 cycles of
30 s with cooling intervals of 1 min on ice, between each cycle.
3. Remove cellular debris by centrifugation at 4000 Â g for
10 min and filter sterilize lysate through 0.2μM filter into a
fresh tube.
4. Store lysates at À20
until needed or proceed with protein
extraction.
Mass Spectrometry-Based Analysis of Mycobacterial Single-Colony Proteome
183