Reviewer
Amit Kumar Dey
  • Biochemist, National Institute on Aging-NIH
Research fields
  • Biochemistry, Molecular Biology, Clinical Proteomics, Mass spectrometry, bottom up proteomics
Muscle Biopsy Sample Preparation and Proteomics Analysis Based on UHPLC-MS/MS
Authors:  Jiawei Du, Jinghua Hou, Hezhang Yun and Yafeng Song, date: 12/20/2024, view: 176, Q&A: 0

Proteomics analysis is crucial for understanding the molecular mechanisms underlying muscle adaptations to different types of exercise, such as concentric and eccentric training. Traditional methods like two-dimensional gel electrophoresis and standard mass spectrometry have been used to analyze muscle protein content and modifications. This protocol details the preparation of muscle samples for proteomics analysis using ultra-high-performance liquid chromatography (UHPLC). It includes steps for muscle biopsy collection, protein extraction, digestion, and UHPLC-based analysis. The UHPLC method offers high-resolution separation of complex protein mixtures, providing more detailed and accurate proteomic profiles compared to conventional techniques. This protocol significantly enhances sensitivity, reproducibility, and efficiency, making it ideal for comprehensive muscle proteomics studies.

Workflow for High-throughput Screening of Enzyme Mutant Libraries Using Matrix-assisted Laser Desorption/Ionization Mass Spectrometry Analysis of Escherichia coli Colonies
Authors:  Kisurb Choe and Jonathan V. Sweedler, date: 11/05/2023, view: 642, Q&A: 0

High-throughput molecular screening of microbial colonies and DNA libraries are critical procedures that enable applications such as directed evolution, functional genomics, microbial identification, and creation of engineered microbial strains to produce high-value molecules. A promising chemical screening approach is the measurement of products directly from microbial colonies via optically guided matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Measuring the compounds from microbial colonies bypasses liquid culture with a screen that takes approximately 5 s per sample. We describe a protocol combining a dedicated informatics pipeline and sample preparation method that can prepare up to 3,000 colonies in under 3 h. The screening protocol starts from colonies grown on Petri dishes and then transferred onto MALDI plates via imprinting. The target plate with the colonies is imaged by a flatbed scanner and the colonies are located via custom software. The target plate is coated with MALDI matrix, MALDI-MS analyzes the colony locations, and data analysis enables the determination of colonies with the desired biochemical properties. This workflow screens thousands of colonies per day without requiring additional automation. The wide chemical coverage and the high sensitivity of MALDI-MS enable diverse screening projects such as modifying enzymes and functional genomics surveys of gene activation/inhibition libraries.


Key features

• Mass spectrometry analyzes a range of compounds from E. coli colonies as a proxy for liquid culture testing enzyme mutant libraries.

• Colonies are transferred to a MALDI target plate by a simple imprinting method.

• The screen compares the ratio among several products or searches for the qualitative presence of specific compounds.

• The protocol requires a MALDI mass spectrometer.


Graphical overview

Overview of the MALDI-MS analysis of microbial colonies for screening mutant libraries. Microbial cells containing a mutant library for enzymes/metabolic pathways are first grown in agar. The colonies are then imprinted onto a MALDI target plate using a filter paper intermediate. An optical image of the MALDI target plate is analyzed by custom software to find the locations of individual colonies and direct subsequent MALDI-MS analyses to the selected colonies. After applying MALDI matrix onto the target plate, MALDI-MS analysis of the colonies is performed. Colonies showing the desired product profiles are found by data analysis via the software, and the colonies are picked for downstream analysis.

Quantification of Chromosomal Aberrations in Mammalian Cells
Authors:  Inés Paniagua and Jacqueline J. L. Jacobs, date: 08/20/2023, view: 1188, Q&A: 0

Maintenance of genome integrity requires efficient and faithful resolution of DNA breaks and DNA replication obstacles. Dysfunctions in any of the processes orchestrating such resolution can lead to chromosomal instability, which appears as numerical and structural chromosome aberrations. Conventional cytogenetics remains as the golden standard method to detect naturally occurring chromosomal aberrations or those resulting from the treatment with genotoxic drugs. However, the success of cytogenetic studies depends on having high-quality chromosome spreads, which has been proven to be particularly challenging. Moreover, a lack of scoring guidelines and standardized methods for treating cells with genotoxic agents contribute to significant variability amongst different studies. Here, we report a simple and effective method for obtaining well-spread chromosomes from mammalian cells for the analysis of chromosomal aberrations. In this method, cells are (1) arrested in metaphase (when chromosome morphology is clearest), (2) swollen in hypotonic solution, (3) fixed before being dropped onto microscope slides, and (4) stained with DNA dyes to visualize the chromosomes. Metaphase chromosomes are then analyzed using high-resolution microscopy. We also provide examples, representative images, and useful guidelines to facilitate the scoring of the different chromosomal aberrations. This method can be used for the diagnosis of genetic diseases, as well as for cancer studies, by identifying chromosomal defects and providing insight into the cellular processes that influence chromosome integrity.


Graphical overview


Bacterial Infection with Listeria monocytogenes in Mice and Subsequent Analysis of Antigen-Specific CD8 T Cell Responses
Authors:  Bojana Jakic, Janine Kimpel, William J. Olson, Verena Labi and Natascha Hermann-Kleiter, date: 12/05/2021, view: 2820, Q&A: 0

Pathogens such as bacteria, viruses, fungi, or protozoa can cause acute and chronic infections in their hosts. The intracellular bacterium Listeria monocytogenes serves as a model pathogen to assess the molecular mechanisms regulating CD8 T cell activation, differentiation, and function. We set up an experimental workflow to investigate cell-intrinsic roles of the nuclear receptor NR2F6 in CD8 T cell memory formation upon Listeria monocytogenes (LmOVA) infection (Jakic et al., 2021). The current protocol details how to cultivate ovalbumin-expressing LmOVA, infect naïve C57BL/6 mice with these bacteria and determine the bacterial load in host organs. Furthermore, we describe how to evaluate antigen-specific CD8 T cell responses and discriminate between short-lived effector and memory precursor cells in vivo following LmOVA infection (Figure 1). To assess CD8 T cell-intrinsic molecular mechanisms, we integrated an adoptive cell transfer (ACT) experiment of genetically modified naïve OT-I CD8 T cells into congenic hosts before LmOVA infection.


Graphic abstract:


Figure 1. Experimental workflow depicting the steps for infection of mice with Listeria and subsequent analysis of antigen-specific CD8 memory responses. Bacteria (ovalbumin expressing Listeria monocytogenes) are thawed and grown on lysogeny broth (LB) plates overnight (ON). A single colony is picked and grown in LB medium ON. Bacteria from the exponential growth phase are then injected into a C57BL/6 mouse via tail vein injection. Colony forming units (CFU) of the bacteria can be detected in the spleen on day 3 post injection. Antigen-specific CD8 T cell immune response can be investigated during the acute phase (d3 after infection), during the peak of the adaptive immune response (d7), the clearance phase (d26), or the memory phase (d70) by flow cytometry. Created with BioRender.com.


Tracing Nitrogen Metabolism in Mouse Tissues with Gas Chromatography-Mass Spectrometry
Authors:  Rong Xu, Yekai Wang and Jianhai Du, date: 02/20/2021, view: 2964, Q&A: 0

Nitrogen-containing metabolites including ammonia, amino acids, and nucleotides, are essential for cell metabolism, growth, and neural transmission. Nitrogen metabolism is tightly coordinated with carbon metabolism in the breakdown and biosynthesis of amino acids and nucleotides. Both nuclear magnetic resonance spectroscopy and mass spectrometry including gas chromatography-mass spectrometry (GC MS) and liquid chromatography (LC MS) have been used to measure nitrogen metabolism. Here we describe a protocol to trace nitrogen metabolism in multiple mouse tissues using 15N-ammonia coupled with GC MS. This protocol includes detailed procedures in tracer injection, tissue preparation, metabolite extraction, GC MS analysis and natural abundance corrections. This protocol will provide a useful tool to study tissue-specific nitrogen in metabolically active tissues such as the retina, brain, liver, and tumor.

Bacterial Adhesion Kinetics in a High Throughput Setting in Seconds-minutes Time Resolution
Authors:  Nimrod Shteindel and Yoram Gerchman, date: 01/20/2021, view: 1976, Q&A: 0
Bacterial surface adhesion, the first step in many important processes including biofilm formation and tissue invasion, is a fast process that occurs on a time scale of seconds. Adhesion patterns tend to be stochastic and spatially heterogeneous, especially when bacteria are present in low population densities and at early stages of adhesion to the surface. Thus, in order to observe this process, a high degree of temporal resolution is needed across a large surface area in a way that allows several replicates to be monitored. Some of the current methods used to measure bacterial adhesion include microscopy, staining-based microtiter assays, spectroscopy, and PCR. Each of these methods has advantages in assaying aspects of bacterial surface adhesion, but none can capture all features of the process. In the protocol presented here, adapted from Shteindel et al., 2019, fluorescently-labeled bacteria are monitored in a multi-titer setting using a standard plate fluorimeter and a dye that absorbs light in the fluorophore excitation and emission wavelengths. The advantage of using this dye is that it restricts the depth of the optic layer to the few microns adjacent to the bottom of the microtiter well, eliminating fluorescence originating from unattached bacteria. Another advantage of this method is that this setting does not require any preparatory steps, which enables reading of the sample to be repeated or continuous. The use of a standard multi-titer well allows easy manipulation and provides flexibility in experimental design.
Extracellular RNA Isolation from Biofilm Matrix of Pseudomonas aeruginosa

Most bacteria in natural ecosystems form biofilms-a bacterial community, surrounded by a polymer matrix that consists mostly of exopolysaccharides, proteins, and nucleic acids. Extracellular RNA as a matrix component is involved in biofilm formation-the fact that was confirmed by direct detection of extracellular RNA in the biofilm matrix, and by an interruption of the biofilm's structure with RNases. Number of protocols describing isolation of RNA from biofilm matrix are limited and usually involve uncommon equipment and reagents. Here we describe simple method for extracellular RNA isolation from biofilm matrix using basic laboratory reagent and equipment. Key steps of the protocol include separation of matrix and bacterial cells with high ionic solution of NaCl, RNA precipitation with LiCl and clean up with option to use inexpensive column for plasmid DNA isolation rather than specialized RNA kits. Described protocol allows to isolate extracellular RNA suitable for further molecular biology procedures such as sequencing, RT-PCR and cloning in less than one day (excluding time for biofilm growing up).

Isolation of Lipid Rafts from Cultured Mammalian Cells and Their Lipidomics Analysis
Authors:  Nigora Mukhamedova, Kevin Huynh, Hann Low, Peter J. Meikle and Dmitri Sviridov, date: 07/05/2020, view: 4270, Q&A: 0
Lipid rafts are distinct liquid-ordered domains of plasma membranes of most eukaryotic cells providing platform for signaling pathways. Lipid composition of rafts is critical for their structural integrity and for regulation of signaling pathways originating from rafts. Here we provide a protocol to isolate lipid rafts from cultured human and animal cells and comprehensively analyse their lipid composition.
Viral Double-Stranded RNA Detection by DNase I and Nuclease S1 digestions in Leishmania parasites
Authors:  Nathalie Isorce and Nicolas Fasel, date: 05/05/2020, view: 3748, Q&A: 1
Many RNA viruses are found in protozoan parasites. They can be responsible for more serious pathology or treatment failure. For the detection of viral double-stranded RNA (dsRNA), sequence-dependent and -independent methods are available, such as quantitative real-time PCR and immunofluorescence, dot blot, ELISA or sequencing. The technique presented here is sequence-independent and is well detailed in the following protocol, taking the example of Leishmania RNA virus (LRV) in Leishmania guyanensis (Lgy) species. To summarise, the protocol is divided into four major steps: RNA extraction from the parasites, RNA purification, enzymatic digestions with DNase I and Nuclease S1, and visualization by gel electrophoresis. This method can be used to detect other viral dsRNA in other parasites. It provides an additional tool, complementary to other techniques previously cited and it is easy and quite fast to achieve.
Determination of Flavin Potential in Proteins by Xanthine/Xanthine Oxidase Method
Authors:  Elena Maklashina and Gary Cecchini, date: 04/05/2020, view: 3670, Q&A: 0
This protocol describes a simple xanthine/xanthine oxidase enzymatic equilibration method for determination of the redox potential of a flavin. As an example of the use of this method, we determine the reduction potential of the covalently bound FAD cofactor (Em = -55 mV) in the SdhA flavoprotein subunit of succinate dehydrogenase from Escherichia coli. In principle, this method can be used routinely to determine the redox potential of flavin cofactors in any simple flavoprotein from equilibrium concentrations with an appropriate reference dye of known Em without the use of sophisticated electrochemical equipment.
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