As the ever-increasing sensitivity of mass spectrometers is allowing the analysis of even more miniscule peptide quantities, our
sample preparation protocols should be developed to allow for
the preparation of smaller quantities of protein. Given the typical
sample loss accrued through sample preparation, [3] in vitro-based
analyses typically rely on large quantities of sample input. While a
detailed proteomic analysis of single bacterial cells is as yet out of
reach, single colony proteomics may be achievable. The study of
individual bacterial colonies will not only push the limits of modern
instruments but also open new avenues in biological research that
would otherwise be out of reach [4].
A shift from bulk culture analysis to single colonies has implications beyond basic research. In a clinical setting, rapid identification
of pathogenic and nonpathogenic bacteria will be very useful in
informing the course of treatment. Single mycobacterial colonies,
for instance, can be studied to assess virulence and drug resistance
factors that may otherwise be lost in liquid media [5]. Here, we
have developed an optimized protocol for the extraction and reproducible quantification of proteomes from very limited samples,
such as single mycobacterial colonies.
2 Materials
All solvents in this protocol were prepared using Milli-Q water with
a resistance of 18.2 MΩ cm and are referred to as “water” in the
protocol.
2.1 Mycobacterium
Smegmatis
This protocol was established using Mycobacterium smegmatis
(strain mc
2 155).
2.2 7H9 Liquid Media
(See Note 1)
1. Middlebrook 7H9 media.
2. Glycerol.
3. Polysorbate 80.
2.3 7H10 Solid Media
(See Note 2)
1. Middlebrook 7H10 media.
2. Glycerol.
3. Agar plates.
2.4 Sterile
Inoculation Loops
1. Disposable sterilized inoculating loops, size-1μL.
2.5 Cell Lysis
1. Lysis buffer (500 mM Tris pH 8, protease and phosphatase
inhibitor cocktail, lysozyme 5μg/mL, 1% sodium dodecyl sulfate, and 1.5% sodium deoxycholate).
2. VirSonic Ultrasonic Cell Disrupter 100.
3. 0.2-μm pore size low-protein binding filters.
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John Iradukunda et al.
sample preparation protocols should be developed to allow for
the preparation of smaller quantities of protein. Given the typical
sample loss accrued through sample preparation, [3] in vitro-based
analyses typically rely on large quantities of sample input. While a
detailed proteomic analysis of single bacterial cells is as yet out of
reach, single colony proteomics may be achievable. The study of
individual bacterial colonies will not only push the limits of modern
instruments but also open new avenues in biological research that
would otherwise be out of reach [4].
A shift from bulk culture analysis to single colonies has implications beyond basic research. In a clinical setting, rapid identification
of pathogenic and nonpathogenic bacteria will be very useful in
informing the course of treatment. Single mycobacterial colonies,
for instance, can be studied to assess virulence and drug resistance
factors that may otherwise be lost in liquid media [5]. Here, we
have developed an optimized protocol for the extraction and reproducible quantification of proteomes from very limited samples,
such as single mycobacterial colonies.
2 Materials
All solvents in this protocol were prepared using Milli-Q water with
a resistance of 18.2 MΩ cm and are referred to as “water” in the
protocol.
2.1 Mycobacterium
Smegmatis
This protocol was established using Mycobacterium smegmatis
(strain mc
2 155).
2.2 7H9 Liquid Media
(See Note 1)
1. Middlebrook 7H9 media.
2. Glycerol.
3. Polysorbate 80.
2.3 7H10 Solid Media
(See Note 2)
1. Middlebrook 7H10 media.
2. Glycerol.
3. Agar plates.
2.4 Sterile
Inoculation Loops
1. Disposable sterilized inoculating loops, size-1μL.
2.5 Cell Lysis
1. Lysis buffer (500 mM Tris pH 8, protease and phosphatase
inhibitor cocktail, lysozyme 5μg/mL, 1% sodium dodecyl sulfate, and 1.5% sodium deoxycholate).
2. VirSonic Ultrasonic Cell Disrupter 100.
3. 0.2-μm pore size low-protein binding filters.
182
John Iradukunda et al.