15. Water for HPLC, LC–MS grade.
16. 98–100% Formic acid.
17. Tryptic hydrolyzate β-galactosidase from the LC/MS peptide/
protein mass standards kit for tuning the ABsciex ESI
instruments.
18. For chromatographic separation, use the following buffer solutions: pre-column sample buffer and Buffer A—98.9% H 2 O,
1% CH 3 CN, 0.1% FA (in volume fractions); Buffer —80%
CH 3 CN, 0.1% FA (in volume fractions).
2.6 Identification
of Mass
Spectrometric Data
1. Mass
spectrometry
data
format
conversion
tool
msConvertGUI.
2. Peptide and protein identification tools MASCOT (version
2.5.1) and X! Tandem (ALANINE, 2017.02.01)—we use
both for thorough protein identification.
3. Scaffold 4 software (version 4.2.1) for visualization and validation of complex MS/MS proteomics experiments.
2.7 Proteogenomic
Analysis
1. R programming language (R Foundation for Statistical Computing), a free software environment for statistical computing
and efficient manipulation with biological data, especially Biostrings library.
3 Methods
3.1 Purification
of Mycobacterial Cells
Mycobacterium tuberculosis is highly pathogenic to humans; therefore, appropriate protective equipment should be used when working with cell culture.
1. Incubate 40 mL of complete 7H9 broth with a loopful of
mycobacteria, vortex to disperse cells, and incubate at 37
C
to mid-log phase (0.6 at OD 600 ). Or cultivate mycobacteria on
a solid media (Lo ¨wenstein–Jensen, Middlebrook agar; single
tube) also to mid-log phase (0.6 at OD 600 ).
2. The mycobacterial cells obtained in the process of cultivation
on solid media should be collected with microbiological loops
in 15 mL Falcon tubes containing 5 mL of wash buffer. Mycobacterial growth in liquid media should be centrifuge at
4,500 Â g for 30 min at 14
C and transfer to 15 mL Falcon
tubes containing 5 mL of wash buffer.
3. Vortex the suspension at full speed for 2–3 min to disrupt the
bacterial colonies. All clumps should be broken up.
4. Heat: inactivate the cells by placing the tube in the oven or
water bath at 80
C for 25 min (seeNote 1).
5. Centrifuge samples at 4,500 Â g for 20 min at 18–20
C.
Proteogenomic Analysis of Mycobacteria
195
16. 98–100% Formic acid.
17. Tryptic hydrolyzate β-galactosidase from the LC/MS peptide/
protein mass standards kit for tuning the ABsciex ESI
instruments.
18. For chromatographic separation, use the following buffer solutions: pre-column sample buffer and Buffer A—98.9% H 2 O,
1% CH 3 CN, 0.1% FA (in volume fractions); Buffer —80%
CH 3 CN, 0.1% FA (in volume fractions).
2.6 Identification
of Mass
Spectrometric Data
1. Mass
spectrometry
data
format
conversion
tool
msConvertGUI.
2. Peptide and protein identification tools MASCOT (version
2.5.1) and X! Tandem (ALANINE, 2017.02.01)—we use
both for thorough protein identification.
3. Scaffold 4 software (version 4.2.1) for visualization and validation of complex MS/MS proteomics experiments.
2.7 Proteogenomic
Analysis
1. R programming language (R Foundation for Statistical Computing), a free software environment for statistical computing
and efficient manipulation with biological data, especially Biostrings library.
3 Methods
3.1 Purification
of Mycobacterial Cells
Mycobacterium tuberculosis is highly pathogenic to humans; therefore, appropriate protective equipment should be used when working with cell culture.
1. Incubate 40 mL of complete 7H9 broth with a loopful of
mycobacteria, vortex to disperse cells, and incubate at 37
C
to mid-log phase (0.6 at OD 600 ). Or cultivate mycobacteria on
a solid media (Lo ¨wenstein–Jensen, Middlebrook agar; single
tube) also to mid-log phase (0.6 at OD 600 ).
2. The mycobacterial cells obtained in the process of cultivation
on solid media should be collected with microbiological loops
in 15 mL Falcon tubes containing 5 mL of wash buffer. Mycobacterial growth in liquid media should be centrifuge at
4,500 Â g for 30 min at 14
C and transfer to 15 mL Falcon
tubes containing 5 mL of wash buffer.
3. Vortex the suspension at full speed for 2–3 min to disrupt the
bacterial colonies. All clumps should be broken up.
4. Heat: inactivate the cells by placing the tube in the oven or
water bath at 80
C for 25 min (seeNote 1).
5. Centrifuge samples at 4,500 Â g for 20 min at 18–20
C.
Proteogenomic Analysis of Mycobacteria
195