Top-Down Approach Overview

By Shelley Farrar Stoakes, M.Sc., B.Sc.Reviewed by Afsaneh Khetrapal, BSc

The top-down approach to systems biology reconstructs metabolic networks through data such as proteomics and transcriptomics.

Credit: VectorFrenzy/Shutterstock.com

It contrasts with the bottom-up approach, which also constructs biological pathway information associated with physiological processes. The top-down approach has historically been utilized in early genome sequencing methodologies and is now being employed in further detailing the structural characterization of nucleic acids.

The field of proteomics is particularly being revolutionized through a top-down approach to create proteoform maps without any loss of information.

Top-down and bottom-up approaches to systems biology

Systems biology is the field of computational modeling of biological systems and has been used to determine the complex macromolecular interactions that occur in an organism.

The metabolic behavior of the cell and the models built to understand it can be approached in two opposite directionalities. The bottom-up approach encompasses draft reconstruction of networks that can be simulated under various physiological conditions.

Data collection of all organism-specific information is assembled into a genome-scale model having been extracted through bioinformatics software tools. In contrast, the top-down approach reconstructs metabolic networks from experimental ‘omics’ data.

Underlying interactions can be understood from the flow of information moving from the proteome or transcriptome to simulated metabolic pathways. The ‘omics’ data required for this approach is produced from common technology such as DNA microarrays and mass spectrometers.

Top-down approach to genome sequencing

An example of a top-down approach can be observed in hierarchical shotgun sequencing. Before being sheared into smaller fragments and reassembled, a low resolution map of the whole genome is produced.  This technique was used in the earliest days of genome sequencing when a minimal amount of high throughput sequencing was required.

By producing a whole genome map first, it is possible to form a series of overlapping fragments, therefore not requiring sequence assembly algorithms. A minimal number of fragments could then be chosen that span the entire chromosome. Historically, the advent of whole genome shotgun sequencing replaced hierarchical shotgun sequencing as the primary method for sequencing genomes, though the Human Genome Project which was completed in 2003 utilized the former method.

Top-down approach to mass spectrometry

A top-down approach to mass spectrometry can be observed in the analysis of intact biomolecules that are a part of a greater complex. Tandem mass spectrometry is being utilized for DNA/RNA identification.

This method has an advantage over conventional biochemical methods of analysis through its ability to determine modifications with greater sensitivity. The fragmented ions identified provide a structural characterization of nucleic acids which can be employed in the development of future gene therapy.  

The top-down approach is also providing greater clarity of modifications to nucleic acids that occur in nature. The addition of modification groups is linked to stability changes and a consequential change in function. By mapping the fragmentation mechanisms of nucleic acid ions, the biomechanism underpinning observed stability changes can be understood and utilized in possible artificial modifications.

Top-down approach to proteomics through mass spectrometry

The most commonly employed top-down approach is in the field of proteomics. Whereas bottom-up mass spectrometry conventionally digests proteins into peptides for identification, top-down proteomics introduces intact proteins to the mass spectrometer with both entire and fragmented ion masses measured.

With the increased understanding of protein complexity, proteoform mapping without any loss of information is crucial. The risk of important details such as sequence variants being lost on separate peptides is avoided through the use of a top-down approach.

Advances to top-down mass spectrometry mean that the mapping of larger proteome complexes is now possible. This has important ramifications for the future of protein therapeutics, particularly the development of larger protein-based drugs that will require tests as a whole and in terms of its molecular components.

Further Reading

• All Mass Spectrometry Content
• What is Mass Spectrometry?
• Mass Spectrometry Applications?
• Using Mass Spectrometry for Protein Complex Analysis
• Coupling and Ionization in Miniature Mass Spectrometry