Proteomics is playing an increasingly important role in the discovery of disease biomarkers and the identification of new therapeutic targets. Proteomics involves the separation, identification, and quantification of proteins. It also includes the characterization and analysis of proteoforms that arise as a result of post-translational modifications (PTMs) and sequence variants such as mutants and alternatively spliced isoforms.
A ball and stick + ribbons models of HIV protease - an enzyme that is the target of much AIDS and HIV research. © lculig / Shutterstock.com
The principal methodology for proteomics is the use of mass spectrometry (MS) in combination with a range of separation methods. The two main approaches to identifying and characterizing proteins using MS are the “bottom-up” and “top-down” methodologies.
Bottom-up and top-down proteomics
In the conventional bottom-up approach, purified proteins or complex mixtures of protein are digested into peptides via proteolytic cleavage, separated (e.g. by a chromatographic column) and the peptide products analyzed in a mass spectrometer.
In top-down proteomics, intact protein ions are generated by electrospray mass spectrometry (ESI), introduced into a mass analyzer and subjected to gas-phase fragmentation. Both approaches are associated with various advantages and limitations.
In bottom-up proteomics, only a small and variable fraction of the total peptide population of a protein can be recovered, resulting in a low percentage coverage of the protein sequence. Genomic studies have shown that each open reading frame can give rise to several protein isoforms, as a result of alternative splicing products and differences in the locations and types of PTMs. The limited sequence coverage in bottom up proteomics means a significant amount of information about PTMs and alternative splice variants is lost.
By contrast, the top-down approach allows MS analysis of intact proteins that have not been cleaved, meaning the labile structural protein characteristics that are mostly destroyed in bottom-up MS are preserved. Universal detection of all existing modifications can be achieved simultaneously in one spectrum and any existing correlations between those modifications can also be determined. In the top-down approach, elimination of protein digestion also results in significant time savings.
Challenges faced in top-down genomics
Top-down proteomics has many advantages over the bottom-down approach and is the superior method for examining protein modification. However, unlike the well-established bottom-up approach, top-down proteomics is still undergoing development and faces many technical challenges before it can be considered as a robust approach for the study of proteomics.
One major challenge is that proteins are generally more complicated to handle than small peptides, due largely to their poor solubility. Unlike the small peptides generated in the bottom-down approach, which are highly soluble in liquid chromatography –MS conditions, proteins may differ in their solubility under the same conditions. Some large proteins and virtually all membrane proteins require a detergent such as sodium dodecyl sulfate (SDS) for their solubilization. However, the presence of an ionic detergent such as SDS is not compatible with the use of ESI and it can therefore not be applied in top-down proteomics.
Artistic impression of a plasma membrane of a human cell. The plasma membrane is a bilayer composed of phopholipids in which lots of transmembrane and surface proteins reside. © somersault1824 / Shutterstock.com
The sensitivity and detection limits of a mass spectrometer are also much poorer for proteins than for peptides. In addition, a protein’s tertiary structure becomes more difficult to disrupt, the greater the protein’s molecular weight, meaning most top-down applications are limited to proteins of less than 50 kDa.
Furthermore, the throughput of top-down proteomics is still not comparable to that of bottom-up shotgun proteomics, although it is rapidly improving. Top-down proteomics has so far mainly been used for the comprehensive characterization of single proteins or only low- to-moderate complexity protein mixtures, with a fairly low throughput.
The future of proteomics
Although top-down proteomics still has some technical challenges to overcome, significant progress has been achieved in this field recently. With further developments, it is hoped that top-down proteomics will become a widely used technique and that synergy between the bottom-up and top-down approaches will eventually result in optimal proteomic findings and broaden the use of proteomics in biomedical research.
Indeed, a melding of the two methods is already in development with the emergence of “middle-down” proteomics. Here, limited proteolytic cleavage of large proteins is performed to yield more manageable polypeptides that can be sequenced using a top-down approach, thereby enabling good sequence coverage and retention of PTM information.
Symposia at Pittcon 2016
This year’s Pittcon, a leading conference and exposition for laboratory science, will take place between the 6th and 10th March 2016 in Atlanta, Georgia.
The event’s impressive technical program will feature more than 2000 presentations. Among these, topics will include the use of proteomics-based approaches as powerful investigative tools for exploring disease mechanisms and potential drug targets.
We will hear about the challenges faced in top-down proteomics and the novel strategies being proposed to address these difficulties including the protein solubility challenge, the proteome complexity challenge and the issue of proteodynamic range.
Six hundred and eighty three exhibitors will attend Pittcon this year and amongst them will be Phytronix Technologies, a leader in quantitative, ultra-fast and high throughput solutions in mass spectrometry. Also exhibiting is Shimadzu Scientific Instruments, a leading provider of unparalleled solutions –based platforms that can address specific laboratory workflows in mass spectrometry applications.
- Ge Y, et al. Comprehensive Analysis of Protein Modifications by Top-Down Mass Spectrometry. Circulation: Cardiovascular Genetics. 2011; 4: 711
- Dalmasso E, et al. Top-down, Bottom-up – The Merging of Two High-Performance technologies. Bioradiations 2009;129
- Wheir T. Top-Down versus Bottom-Up Approaches in Proteomics. LC-GC chromatographyonline.com 2006: Issue 9
- Chait B. Mass Spectrometry: Bottom-Up or Top-Down? Science 2006;314:65–66
Pittcon® is a registered trademark of The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, a Pennsylvania non-profit organization. Co-sponsored by the Spectroscopy Society of Pittsburgh and the Society for Analytical Chemists of Pittsburgh, Pittcon is the premier annual conference and exposition on laboratory science.
Proceeds from Pittcon fund science education and outreach at all levels, kindergarten through adult. Pittcon donates more than a million dollars a year to provide financial and administrative support for various science outreach activities including science equipment grants, research grants, scholarships and internships for students, awards to teachers and professors, and grants to public science centers, libraries and museums.
Visit pittcon.org for more information.
Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.