Proteins can be subjected to a wide variety of targeted post-translational modifications that will considerably modulate their function. Fortunately, several new technologies have emerged to assist in identification and analysis of these modifications, shedding new light on an important layer of proteomic complexity. Caitlin Smith reports.
A growing but extremely important subsector of the field of proteomics involves the study of post-translational modification (PTM), the process by which proteins are enzymatically appended with a wide variety of chemical groups, including lipids, carbohydrate groups and even other proteins. The addition and removal of these groups can dramatically modulate the function of a given protein, and PTMs are known to be involved in the mediation of a staggering variety of processes relating to cell signaling and gene expression, among others.
Unfortunately, the systematic analysis of PTMs is no mean feat. Roland Annan, head of the Proteomics and Biological Mass Spectrometry Group at GlaxoSmithKline, says that one of the biggest obstacles is “the incredible diversity of post-translational modifications and amazing number of different ones that you can find on one gene product. For this reason, I think many individual proteins are their own little proteomes, complex and highly regulated.” Although some of the most well-known PTMs include phosphorylation of serine, threonine and tyrosine residues of proteins, there are many other important modifications that also are highly regulated and have a crucial function in cell signaling. Examples of these include acetylation, acylation, glycosylation, methylation, sulfation and ubiquitination, among others. Much of recent research has also focused on histones, where PTMs are thought to be central for dictating the structure of chromatin, affecting cellular proliferation and development.
As daunting as the challenge may seem, however, there are useful tools available to the PTM investigator. “Mass spectrometry has long been a powerful technique to identify post-translational modifications and will continue to be one of the primary methods,” says John Yates, a faculty member in cell biology at the Scripps Research Institute. According to Yates, “recent improvements in mass spectrometers (for example, high resolution and high mass accuracy) and new ion dissociation methods have been shown to help identify new types of modifications. Improved resolution and mass accuracy help to differentiate modifications, such as tyrosine phosphorylation and sulfation.” These ongoing developments in mass spectroscopy and associated technologies are now allowing PTM researchers to get more quantitative results and discover more than ever before.
Putting PTMs on the map
Before mass spectroscopy can be applied to the mapping of PTMs within proteins, the protein of interest must be prepared as an appropriate sample for mass spectroscopy analysis. For this 'bottom-up approach' to proteomics, the first step in the preparation process is purification, for which there are a variety of methods. Gel electrophoresis remains a workhorse favorite, with two-dimensional gel electrophoresis being particularly useful for mass spectroscopy preparation. “Two-dimensional electrophoresis remains a method of choice for evidencing post-translational modifications, and also separating modified proteins from unmodified ones, which increases dramatically the probability of finding the modified peptides,” says Thierry Rabilloud, of the Laboratory of Cell Bioenergetics, of the French Atomic Energy Commission. “This is very important when an undirected modification analysis (that is, without any prior hypothesis) is to be performed.” Several companies, including Bio-Rad, Topac, Pierce, Sigma-Aldrich and G Biosciences, offer materials for two-dimensional gel electrophoresis. In addition, Nextgen Sciences offers equipment and reagents based on their a2DEoptimizer system.
Another commonly used method is antibody-based affinity purification. Companies are now marketing antibodies directed at all proteins that have a given PTM, such as anti-methyl or anti-phosphotyrosine antibodies. Although there is some question about the effectiveness of these 'pan-PTM' antibodies, they can be useful for a first-pass purification step. Companies offering these antibodies include AbCam, Upstate and Invitrogen.
Once you have a relatively purified sample of protein containing your PTM(s) of interest, the next step involves the chemical or enzymatic degradation of the protein and then separation of the peptides. The latter is usually done by high-performance liquid chromatography (HPLC) and mass spectroscopy (MS) to separate the peptide fragments according to their masses. The mass spectra often show patterns that belie particular PTMs—for example, multiple mass shifts of 162 kDa for glycosylation sites, and 'satellite' masses of 98 kDa for phosphoserine and phosphothreonine modifications that result from losing phosphoric acid groups.
Advion Biosciences is trying to speed this process with their TriVersa NanoMate, a system integrating HPLC fraction collection and MS in one platform. In the same vein, Agilent Technologies is aiming for user-friendly performance in their new Agilent 6210 TOF LC/MS mass spectrometer, which can analyze complex mixtures of proteins with greater accuracy and speed than its predecessor. Not to be outdone, Applied Biosystems recently unveiled its QSTAR Elite LC/MS/MS System, which is billed as fast, accurate and flexible, with the ability to incorporate different types of liquid chromatography workflows.
“The most reliable approach to localize a post-translational modification in the primary structure of a protein or peptide is the recording of tandem MS spectra,” says Albert Sickmann, group leader of the Protein Mass Spectrometry & Functional Proteomics Group at the University of Würzburg. “The interpretation of tandem mass spectra allows the localization of certain mass differences directly to amino acid side chains.” In tandem mass spectroscopy, or MS/MS, the fragments are put through another round of MS, in which the peptides themselves are fragmented by collision with an inert gas. Despite the fact that this process can break peptide bonds, some modified amino acids remain intact—another reason that mass spectroscopy techniques are invaluable to PTM researchers, whose subjects can be quite labile (Box 1). “Future developments have to focus on the detection of more labile (for example, glycans, fatty acids) or nearly isobaric (for example, phosphorylation-sulfation and tri-methylation–acetylation) post-translational modifications,” comments Sickmann. Further advances on the horizon, however, will make these labile modifications more amenable to study.
New alternatives for MS
Advances in mass spectroscopy using the 'top-down' approach to proteomics, in which intact proteins are ionized directly without previous peptide fragmentation, are proving especially fruitful for PTM analysis, yielding greater mass accuracy than ever before. One such approach is electron capture dissociation (ECD), a new method that cleaves proteins between their backbone amide and alpha carbon. A major advantage of ECD is that labile PTMs like phosphorylation, O-glycosylation, and N-glycosylation remain attached to the protein backbones, allowing determination of the site and identity of PTMs. Alan Marshall, professor and director of the Ion Cyclotron Resonance Program at Florida State University, describes ECD as “most generally reliable for locating the site of modification without cleaving off the modifying group.” This ability to preserve PTMs is making ECD a tool of choice for generating high-quality data. Bruker Daltonics recently became the first to offer a commercial instrument for electron transfer dissociation, an alternative method for protein fragmentation that also preserves PTMs. Their HCTultra ion trap facilitates the identification of the types and locations of various PTMs, and is incorporated into their HCTultra PTM Discovery System, which they bill as ideal for researching labile PTMs.
Combining new methods in mass spectroscopy is also a recurring theme in PTM research. For example, Marshall says that combining ECD with other methods, such as infrared multiphoton dissociation or collision-induced dissociation, is especially effective “for determining the branching pattern in glycans [because] ECD cleaves the primary chain, but the two fragments adhere to each other until heated by [infrared radiation].” Roman Zubarev, a professor at Uppsala University, says that a combination of methods is necessary to fragment proteins of interest to the desired degree: “In my opinion, the bottleneck is the absence of high-quality fragmentation techniques. Collisional activation alone does not deliver full sequence information. A combination of complementary fragmentation techniques will be required, such as collisional activation–electron capture–transfer dissociation or 157 nm [ultraviolet light] photodissociation.” Other experts advocate combining 'bottom-up' with 'top-down' approaches for the greatest effectiveness. “In order to be certain,” says Shao-En Ong, a scientist at the Broad Institute of MIT and Harvard, “methods combining some fractionation (like liquid chromatography) and very high mass accuracy instruments like the Fourier transform mass spectrometer, combined with rich sequence information from fragmentation experiments (such as ECD fragmentation techniques) are very useful. Instruments that can combine both these elements are therefore certain to be workhorses for PTM, and indeed, proteomic analysis.”
Many proteomics researchers agree that data management is a challenge to their field. Marshall says that big improvements are needed in “the development of a single standardized publicly available platform for data registry, searching, analysis and reporting” for PTMs (Box 2). Andreas Schlosser, a scientist at the Institute of Medical Immunology in Berlin, agrees: “Perhaps the main challenge is to find ways to handle the enormous complexity and amount of data.”
Another challenge is the ability to study the PTMs in complex mixtures of proteins. “I think the real difficulty we'll have is how we can study the combination of different PTMs in a single sample,” says Ong. “So much of what we know today is based on enrichment from the biological sample. Such protocols are designed to enrich for specific classes of PTM but could potentially miss other modified peptides.”
Containing the cost of analysis will also be a major challenge to analyzing PTMs in human samples, according to Zubarev. “The cost of high-quality analysis of one sample is on the order of 100,000 Euro [US $120,000]. With such a cost, complete PTM analysis for the human proteome is unrealistically expensive. The cost needs to be reduced by a factor of 100 to put the above task into a realistic budget. Mass spectrometry is progressing very fast, but there is a lot to be done before we even start to approach the possibility of large-scale PTM analysis in human samples.” He concludes that “high-resolution methods are the only methods that will survive in the long run. The quest for obtaining full sequence information (which is especially important for PTM analysis) has only started.” (See Table 1)