Chapter 11
Mass Spectrometry-Based Analysis of Mycobacterial
Single-Colony Proteome
John Iradukunda, Tariq Ganief, Jonathan M. Blackburn,
and Nelson C. Soares
Abstract
Mass spectrometry-based single-cell proteomic analysis has recently gained momentum and is now an
emerging area with established protocols and promising results. Traditional proteomic studies, especially
involving bacteria, have been limited to suspension cultures with large protein yields. Such studies,
however, remain population centered with the uniqueness of individual responses to environmental
challenges becoming diluted. To enable bacterial single-colony proteomics, we describe a quantitative
mass spectrometry-based protocol to isolate and analyze the proteome of a single mycobacterial colony
from 7H10 media, with growth supplements for optimal growth. Following protein purification and
digestion, tryptic peptides are analyzed by UHPLC coupled to a hybrid Q Exactive mass spectrometer.
Raw data were analyzed using the MaxQuant Suite, and downstream statistical analysis was performed using
Perseus software. A total of 7805 unique peptides and 1387 proteins were identified. Data are available via
ProteomeXchange with identifier PXD018168. In this chapter, we identify steps most prone to sample loss
and describe measures of alleviation that allows the preservation of protein yield and boosts quantitative
power while increasing reproducibility, of “very limiting samples.”
Key words Single-colony, Proteomics, Mycobacteria, Mass spectrometry, Protein extraction, Bacterial proteome
1 Introduction
The most commonly implemented bacterial bottom-up proteomics
methods are based on proteins extracted from cells grown in liquid
media leading to bulk protein yields. However, these bulk cultures
and subsequent protein harvests belie the notion that heterogeneity
of a population increases over time and leads to heterogeneous
responses within the population. Intrinsic heterogeneity within a
culture population can shed light on global phenomena that are
lineage specific. An example of this can be notably seen in minimal
residual disease, where adaptive evolution plays a major role in
limiting the efficacy of antibiotic treatment [1, 2].
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_11, © Springer Science+Business Media, LLC, part of Springer Nature 2021
181
Mass Spectrometry-Based Analysis of Mycobacterial
Single-Colony Proteome
John Iradukunda, Tariq Ganief, Jonathan M. Blackburn,
and Nelson C. Soares
Abstract
Mass spectrometry-based single-cell proteomic analysis has recently gained momentum and is now an
emerging area with established protocols and promising results. Traditional proteomic studies, especially
involving bacteria, have been limited to suspension cultures with large protein yields. Such studies,
however, remain population centered with the uniqueness of individual responses to environmental
challenges becoming diluted. To enable bacterial single-colony proteomics, we describe a quantitative
mass spectrometry-based protocol to isolate and analyze the proteome of a single mycobacterial colony
from 7H10 media, with growth supplements for optimal growth. Following protein purification and
digestion, tryptic peptides are analyzed by UHPLC coupled to a hybrid Q Exactive mass spectrometer.
Raw data were analyzed using the MaxQuant Suite, and downstream statistical analysis was performed using
Perseus software. A total of 7805 unique peptides and 1387 proteins were identified. Data are available via
ProteomeXchange with identifier PXD018168. In this chapter, we identify steps most prone to sample loss
and describe measures of alleviation that allows the preservation of protein yield and boosts quantitative
power while increasing reproducibility, of “very limiting samples.”
Key words Single-colony, Proteomics, Mycobacteria, Mass spectrometry, Protein extraction, Bacterial proteome
1 Introduction
The most commonly implemented bacterial bottom-up proteomics
methods are based on proteins extracted from cells grown in liquid
media leading to bulk protein yields. However, these bulk cultures
and subsequent protein harvests belie the notion that heterogeneity
of a population increases over time and leads to heterogeneous
responses within the population. Intrinsic heterogeneity within a
culture population can shed light on global phenomena that are
lineage specific. An example of this can be notably seen in minimal
residual disease, where adaptive evolution plays a major role in
limiting the efficacy of antibiotic treatment [1, 2].
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_11, © Springer Science+Business Media, LLC, part of Springer Nature 2021
181