Having grown beyond early ‘proof-of-principle’ studies, top-down proteomics is now extensively used to analyze intact proteins in numerous applications. Here, we discuss how the top-down proteomics approach emerged, the reasons underpinning its success, and highlight some of the most exciting current developments.
The evolution of “bottom-up” approaches
Historically, when someone wanted to decipher the sequence of a protein, he or she would turn to good old wet chemistry, such as the Edman degradation technique. To quantify the amount of that specific protein in a biological sample, they would rely on antibody-based methods; for example, a quantitative western blot or an ELISA assay. To then analyze protein isoforms, intact proteins or protein complexes, either two-dimensional polyacrylamide gel electrophoresis, native gel electrophoresis or size-exclusion chromatography would be employed. Many of these methods are alive and well, partly because one single technique is unlikely to ever fully resolve and characterize certain proteins. However, fast-forward a couple of decades and the technological advancements in chromatography, mass spectrometry (MS) and bioinformatics have put MS-based proteomics as a clear favourite to perform most of these analytical tasks.
The field of proteomics has mainly evolved around “bottom-up” techniques.
In bottom-up experiments, intact proteins are extracted from a cell or tissue lysate and proteolytically digested into peptides. The digestion is usually achieved with a sequence-specific enzyme, for example trypsin or endoproteinase Lys-C. Next, the mixture is separated using liquid chromatography and introduced into the mass spectrometer for MS/MS analysis. Once inside the instrument, the peptides are bombarded with molecules of neutral gas and broken down into smaller fragments, a process known as collision-induced dissociation (although other fragmentation techniques are also in use). To then infer the sequences of the original proteins present in the sample, peptide MS/MS spectra are compared to theoretical fragmentation patterns derived from in silico digested protein databases.
“The reason we use bottom-up approaches, sometimes also called “shotgun” proteomics, is that we can routinely perform very robust and reproducible identification and quantification analyses,” says Ole N. Jensen, a Professor of Protein Mass Spectrometry at the Department of Biochemistry and Molecular Biology; University of Southern Denmark in Odense.
If done correctly the approach is speedy and affords high proteome coverage with good detection limits. “Another reason we use bottom-up is that trypsin generates beautiful peptides that are amenable to mass spectrometry, and we do get very high sensitivity in our experiments,” adds Professor Jensen.
A definition of top-down proteomics
However, despite the many benefits of bottom-up, we still need other methods. “We know that proteins are almost never modified with just one post-translational modification [PTM],” explains Simone Sidoli, a former member of Jensen’s group and now a postdoctoral researcher at the Perelman School of Medicine, University of Pennsylvania, USA. “And when it comes to identifying co-existing PTMs, for example in order to unambiguously determine the histone variants on which certain PTMs reside, shotgun MS approaches can struggle”.
“Top-down” is the term used to describe an alternative proteomics approach, where proteins are not enzymatically digested into peptides. They are introduced into the mass spectrometer in their intact state instead. These intact proteins are then directly analyzed, before undergoing fragmentation. The process decreases initial sample complexity, and crucially preserves the connections between PTMs, sequence variations, alternative splicing, or any other features originally present in each of the intact protein molecules that are being analyzed. Underpinning the success of top-down proteomics are the developments of both Fourier-transform-based, and quadrupole time-of-flight-based high-resolution MS. Equally, the introduction of efficient fragmentation methods, such as electron-transfer dissociation and higher-energy collisional dissociation has been key.
Scientists can now access information that would be unavailable in a standard shotgun experiment, allowing them to i) interrogate structures of large proteins, ii) map combinations of PTMs with full sequence coverage, and iii) identify and measure proteoform distributions.
Making a mark: reading histone modifications using top-down
As with many new techniques, early applications of top-down proteomics were primarily concerned with demonstrating that the platform could work as intended and be applied to answer important biological questions. Not surprisingly, as an ideal tool for mapping coexisting PTMs, the technique achieved success in the field of chromatin biology and characterization of histone PTMs.
Histones are small, positively charged proteins that help package chromosomal DNA inside the cell’s nucleus. “My personal perspective is that we should not consider histones as proteins that “do” things; they are not enzymes. However, that does not imply they are less important,” says Dr. Sidoli. “Based on how histones are assembled and modified, they define chromatin structure, and chromatin is the control panel of a cell.” Importantly, a plethora of chemical alterations on the histone amino (N)-terminal tails regulate chromatin structure and stability, and in effect many aspects of gene expression. Some of the most studied modifications include acetylation, methylation and phosphorylation. Accumulating evidence suggests that various proteases are able to clip the histone tails, leading to an additional layer of regulation.
Professor Jensen and his group in Denmark have been studying these modifications for the past 10 years. In one study, published in the journal Molecular & Cellular Proteomics, they used top-down methods to show how intact and clipped human histones are decorated with different PTM patterns. They first cultivated a human hepatocellular carcinoma cell line in two different cultures; standard two-dimensional (2D) cell monolayers, and three-dimensional (3D) spheroid cultures designed to better mimic the in vivo cell environment. They found that histones grown in 3D culture were clipped, while those grown in 2D stayed intact. Using their top-down strategy, they were then able to identify the sites where clipping takes place. Further MS analysis revealed differences between the relative abundances of a series of PTMs on intact and clipped histone proteoforms.
“That was a new discovery. People have previously shown that histones are clipped at distinct sites. But here we showed that PTMs, mainly methylation status, also differ in these circumstances,” says Prof. Jensen, “this is exciting, as it points towards a regulatory mechanism.”
Structural characterization of monoclonal antibodies
Top-down analysis has also become an important tool for the characterization of biologics, i.e. pharmaceutical drugs produced in living cells. “The beauty of the top-down approach is that in principle you could obtain all modifications on that one intact protein you are most interested in. It works particularly well if you have large quantities of a purified protein. And that is what the pharmaceutical industry does all the time when they work with biopharmaceutical proteins,” says Prof. Jensen.
Indeed, an increasing number of antibodies, growth factors, and interleukins are being produced and brought to market. These molecules are significantly more complex than traditional small organic compounds. The depth and quality of their characterization must be higher for that reason alone. In addition, variations in proteoforms produced during the manufacturing process can affect the efficacy and safety of such drugs. Complete characterization is required to fulfill regulatory standards.
Take recombinant monoclonal antibodies (mAbs) for example. These large, Y-shaped glycoproteins that bind target antigens with high specificity are important in therapeutics targeting cancer, autoimmune disorders and infectious diseases. The characterization of their subunits by means of top-down proteomics has become an important analytical technique for sequence validation or determination of glycan modifications.
A tool for the future
Despite great progress in recent years, some challenges remain for top-down proteomics. The technique is relatively low-throughput as compared to bottom-up experiments, partly because of difficulties in automated liquid chromatography separation of intact proteins. However, useful alternatives are in development. A recent study reported identification of nearly six thousand proteoforms from the E. coli proteome using capillary zone electrophoresis coupled with a commercially available Fourier-transform-based mass spectrometer.
Overall, the field looks set to expand its reach and become an indispensable research tool in the future. “Top-down is going to quickly develop, but it’s not going to happen overnight,” says Professor Jensen, “I think in the next five to ten years we are going to see huge advances.”