6. 0.5 M dithiothreitol (DTT): prepared in 50 mM ammonium
bicarbonate (see Note 12).
7. 0.55 M iodoacetamide (IAA): prepared in 50 mM ammonium
bicarbonate (see Note 13).
8. Acetone (stored at À20
C).
9. Acetonitrile, LC-MS grade.
10. C18 spin columns or C18 desalting tips.
11. ProteaseMax surfactant solution: For a 1% solution, reconstitute 1 mg of ProteaseMax in 100 μL of 50 mM ammonium
bicarbonate. To make aliquots of a 0.2% ProteaseMax solution,
add 4 μL of the 1% solution to 16 μL of 50 mM ammonium
bicarbonate per sample (see Note 14).
12. Modified trypsin sequencing grade: 0.1 mg/mL in 50 mM
ammonium bicarbonate (see Notes 8 and 9).
13. Trifluoroacetic acid (TFA).
14. Bath sonicator.
15. Water bath, hot plate, or incubator at 50
C.
16. Shaker.
17. Microcentrifuge.
18. Vacuum concentrator.
3 Methods
Protein fractions isolated from live M. tuberculosis must be sterile
filtered, heated, or treated with organic solvent (greater than 20%
vol:vol) such as chloroform:methanol 2:1 v/v prior to use outside
of a BSL3 laboratory.
3.1 Processing Intact
Proteins for Top Down
Proteomics Using
a MALDI-ToF Mass
Spectrometer (See
Note 15)
Concentrated protein samples should be quantified using bicinchoninic acid or similar assay as per manufacturer’s instructions to
normalize data intensities. Dilute protein samples may be analyzed
without quantification.
1. Make a fresh MALDI matrix solution.
2. Spot sample on the MALDI target plate: pipette 1 μL of sample
on the plate first (otherwise, the matrix might dry before the
mixing occurs).
3. Pipette 1 μL of the matrix solution on top of the sample and
mix well by pipetting up and down.
4. Allow the mixture to air-dry (see Note 16).
5. Spot the calibration standard on the MALDI target plate by
pipetting 1 μL of standard on the plate first (as above).
538
Carolina Mehaffy et al.
bicarbonate (see Note 12).
7. 0.55 M iodoacetamide (IAA): prepared in 50 mM ammonium
bicarbonate (see Note 13).
8. Acetone (stored at À20
C).
9. Acetonitrile, LC-MS grade.
10. C18 spin columns or C18 desalting tips.
11. ProteaseMax surfactant solution: For a 1% solution, reconstitute 1 mg of ProteaseMax in 100 μL of 50 mM ammonium
bicarbonate. To make aliquots of a 0.2% ProteaseMax solution,
add 4 μL of the 1% solution to 16 μL of 50 mM ammonium
bicarbonate per sample (see Note 14).
12. Modified trypsin sequencing grade: 0.1 mg/mL in 50 mM
ammonium bicarbonate (see Notes 8 and 9).
13. Trifluoroacetic acid (TFA).
14. Bath sonicator.
15. Water bath, hot plate, or incubator at 50
C.
16. Shaker.
17. Microcentrifuge.
18. Vacuum concentrator.
3 Methods
Protein fractions isolated from live M. tuberculosis must be sterile
filtered, heated, or treated with organic solvent (greater than 20%
vol:vol) such as chloroform:methanol 2:1 v/v prior to use outside
of a BSL3 laboratory.
3.1 Processing Intact
Proteins for Top Down
Proteomics Using
a MALDI-ToF Mass
Spectrometer (See
Note 15)
Concentrated protein samples should be quantified using bicinchoninic acid or similar assay as per manufacturer’s instructions to
normalize data intensities. Dilute protein samples may be analyzed
without quantification.
1. Make a fresh MALDI matrix solution.
2. Spot sample on the MALDI target plate: pipette 1 μL of sample
on the plate first (otherwise, the matrix might dry before the
mixing occurs).
3. Pipette 1 μL of the matrix solution on top of the sample and
mix well by pipetting up and down.
4. Allow the mixture to air-dry (see Note 16).
5. Spot the calibration standard on the MALDI target plate by
pipetting 1 μL of standard on the plate first (as above).
538
Carolina Mehaffy et al.