Chapter 23
Methods for Proteomic Analyses of Mycobacteria
Carolina Mehaffy, Megan Lucas, Nicole A. Kruh-Garcia,
and Karen M. Dobos
Abstract
The use of proteomic technologies to characterize and study the proteome of mycobacteria has provided
important information in terms of function, diversity, protein–protein interactions, and host-pathogen
interactions in Mycobacterium spp. There are many different mass spectrometry methodologies that can be
applied to proteomics studies of mycobacteria and microorganisms in general. Sample processing and
appropriate study design are critical to generating high-quality data regardless of the mass spectrometry
method applied. Appropriate study design relies on statistical rigor and data curation using bioinformatics
approaches that are widely applicable regardless of the organism or system studied. Sample processing, on
the other hand, is often a niched process specific to the physiology of the organism or system under
investigation. Therefore, in this chapter, we will provide protocols for processing mycobacterial protein
samples for the specific application of Top-down and Bottom-up proteomic analyses.
Key words Sample processing, Top-Down Proteomics, Bottom-Up Proteomics, Shotgun proteomics, PTM (Post-translational modification), MRM (Multiple Reaction Monitoring), LC-MS (Liquid
Chromatography - Mass Spectrometry), ESI (Electro-Spray Ionization), MALDI-ToF (Matrix
Assisted Laser Desorption Ionization - Time of Flight)
1 Introduction
Proteomics represents a compilation of mass spectrometry-based
techniques that allow for the characterization and global profiling
of proteins (i.e., identification, quantification, and posttranslational
modifications), as reviewed by Jean et al. [1]. Proteomic studies can
be used to gain insight into the physiology of a particular organism,
understand cellular response to stresses, characterize hostpathogen interactions, and identify biomarkers of disease, among
others. Shotgun proteomics, also known as untargeted proteomics
or bottom-up proteomics, is used to comprehensively identify and
quantify proteins in a complex biological sample (i.e., whole cell
lysates, subcellular fractions, and culture filtrate proteins). Shotgun
proteomics have been extensively used to study mycobacterial
pathogens and disease and have resulted in an increased
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_23, © Springer Science+Business Media, LLC, part of Springer Nature 2021
533
Methods for Proteomic Analyses of Mycobacteria
Carolina Mehaffy, Megan Lucas, Nicole A. Kruh-Garcia,
and Karen M. Dobos
Abstract
The use of proteomic technologies to characterize and study the proteome of mycobacteria has provided
important information in terms of function, diversity, protein–protein interactions, and host-pathogen
interactions in Mycobacterium spp. There are many different mass spectrometry methodologies that can be
applied to proteomics studies of mycobacteria and microorganisms in general. Sample processing and
appropriate study design are critical to generating high-quality data regardless of the mass spectrometry
method applied. Appropriate study design relies on statistical rigor and data curation using bioinformatics
approaches that are widely applicable regardless of the organism or system studied. Sample processing, on
the other hand, is often a niched process specific to the physiology of the organism or system under
investigation. Therefore, in this chapter, we will provide protocols for processing mycobacterial protein
samples for the specific application of Top-down and Bottom-up proteomic analyses.
Key words Sample processing, Top-Down Proteomics, Bottom-Up Proteomics, Shotgun proteomics, PTM (Post-translational modification), MRM (Multiple Reaction Monitoring), LC-MS (Liquid
Chromatography - Mass Spectrometry), ESI (Electro-Spray Ionization), MALDI-ToF (Matrix
Assisted Laser Desorption Ionization - Time of Flight)
1 Introduction
Proteomics represents a compilation of mass spectrometry-based
techniques that allow for the characterization and global profiling
of proteins (i.e., identification, quantification, and posttranslational
modifications), as reviewed by Jean et al. [1]. Proteomic studies can
be used to gain insight into the physiology of a particular organism,
understand cellular response to stresses, characterize hostpathogen interactions, and identify biomarkers of disease, among
others. Shotgun proteomics, also known as untargeted proteomics
or bottom-up proteomics, is used to comprehensively identify and
quantify proteins in a complex biological sample (i.e., whole cell
lysates, subcellular fractions, and culture filtrate proteins). Shotgun
proteomics have been extensively used to study mycobacterial
pathogens and disease and have resulted in an increased
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_23, © Springer Science+Business Media, LLC, part of Springer Nature 2021
533