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Proteomics research focuses on the sequencing of intact proteins to gain a deeper understanding of how modifications bring about different changes in protein structure and function. Proteoforms—the protein variants produced from any given gene—have a different biological function. Post-translational modifications (PTMs), such as glycosylation and phosphorylation, may change the nature of protein behavior and form an important association with numerous human diseases. Researchers in many fields are thus eager to characterize these relationships.
Understanding protein structure–function relationships by mapping specific variants to their associated biological activity can be done using mass spectrometry. High-resolution MS is often used in proteomics research to help researchers better understand the molecular and cellular mechanisms of disease. Bottom-up proteomics is the most widely used technique, but does not provide the holistic view of protein structure achieved using the top-down approach. This alternative method directly analyzes intact proteins and, by maintaining their structural integrity, MS-based top-down proteomics enables the mapping of PTMs with full sequence coverage, as well as quantification of specific proteoforms. It is through this quantification that the biological relevance of MS data is improved and proteoforms can be probed for disease biomarkers.
Top-down proteomics case study
Professor Ying Ge’s research lab (http://crb.wisc.edu/yinglab), Department of Chemistry at the University of Wisconsin, Madison, uses MS-based top-down proteomics technology to investigate the molecular and cellular mechanisms underlying cardiovascular diseases. The laboratory focuses on this high-resolution approach over bottom-up proteomics to obtain the most powerful data possible. As a leader in the field, Professor Ge’s aim is to use sophisticated instrumentation to propel top-down proteomics into the realm of mainstream science.
According to Professor Ge, “One of the previous challenges with the top-down approach was the instrument’s capabilities, but now we are in the best position to integrate these technologies with biology and medicine. At the moment, top-down proteomics is still challenging, and most mass spectrometers are designed for bottom-up. With the availability of these new instruments specially tailored for top-down, my lab can present solutions to address the challenges, and help the community adapt to the top-down methods.”
Despite its power as an analytical technique, top-down proteomics is yet to be implemented by most routine labs. Before this method becomes fully robust, protein solubility must be addressed. Professor Ge’s lab is developing novel top-down mass spectrometry-compatible surfactants that can effectively solubilize all protein categories. Sample preparation is another issue that prevents adoption of the technique, but Professor Ge is committed to helping the proteomics community overcome these limitations. “In comparison to the mass spectrometry instruments 10 years ago, those available today are phenomenal. There’s a day-and-night difference between their capabilities for high-resolution data acquisition. It’s now very likely that more and more proteomics labs will adapt to the top-down approach soon, as the advancements make it so much more accessible,” she said.
The Ge laboratory uses the solariX XR (Fourier transform MS or FTMS; XR indicates extreme resolution), Maxis II, and Impact II (Q-TOF) from Bruker Daltonics (Billerica, MA). Professor Ge used FTMS due to its unparalleled resolution and high sensitivity, making it the most sophisticated technique for characterizing large proteins through a top-down approach. Of the new generation of mass spectrometers, she said, “[W]e use Q-TOF for more routine analysis and high-throughput proteomics, which you can do on a daily basis—unimaginable compared to 10 years ago. These instruments have hardly any downtime; they essentially work 24/7, which of course enables us to obtain huge amounts of data. With such advancements in technology, the major bottlenecks are now being overcome.”
Professor Ge’s focus on proteomics stems from the need to better understand how molecules interact as a system, in order to elucidate cellular system functions in health and disease. The Q-TOF instruments have sophisticated proteoform profiling capabilities that allow intact protein masses to be measured from high-complexity protein mixtures to facilitate disease biomarker discovery, while FTMS provides the resolution necessary to resolve large proteins to ensure the success of top-down proteomics.
Future of proteomics
Proteomics is ideal for use in biomarker discovery because numerous molecules are investigated simultaneously. The proteome reflects the dynamic interactions between genes and the environment and is most likely to be affected by disease,1 and therefore lends itself to this particular application. New research into top-down phosphoproteomics has uncovered a method for integrating phosphoprotein enrichment and online LC-MS/MS to enrich, quantify, and characterize phosphoproteins from complex protein mixtures.2 Additional research into front-end intact protein separation, MS detection of large proteins, and MS/MS fragmentation techniques is required before top-down becomes a fully robust method for comprehensive phosphoproteosome analysis.
The extreme resolution and high accuracy of MS instruments power top-down proteomics and fill the gaps left by bottom-up techniques. MS-based top-down proteomics can handle the molecular complexity of proteins, quantify proteoforms, deep-sequence intact proteins, discover unexpected modifications, quantify positional isomers, and determine the order of multiple modifications.
Rapid developments in mass spectrometry technology facilitate innovative solutions to the challenges in top-down proteomics. Most current mass spectrometers are intended for bottom-up proteomics, but collaboration between researchers and vendors to create new instruments for top-down purposes will continue to advance this novel approach.
References
- Horgan, R.P. and Kenny, L.C. “’Omic” technologies: genomics, transcriptomics, proteomics and metabolomics. The Obstetrician and Gynecologist 2011, 13, 189–95.
- Chen, B.; Hwang, L. et al. Coupling functionalized cobalt ferrite nanoparticle with online LC/MS/MS for top-down phosphoproteomics. Chem. Sci. 2017; doi 10.1039/C6SC05435H.
Additional reading
http://pubs.acs.org/doi/abs/10.1021/acs.analchem.7b00380
Rohan Thakur is executive vice president at Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany; e-mail: [email protected]; www.bruker.com