A Shotgun Approach

Dr Shabaz Mohammed, from Utrecht University in the Netherlands, spoke to LCGC about his group’s activities in protein analysis and the importance of “shotgun proteomics.”

Q: Why are you interested in researching proteins?

A: Proteins represent a major class of biomolecules that aid in the functioning of cells. Their activity is modulated by changes to their structure that are performed by the chemical modification of various amino acids. Such changes are often referred to as post‑translational modifications or PTMs. Each cell contains over 10, 000 proteins spanning seven orders of magnitude in abundance: the proteome. Each protein can be present in multiple forms because of these PTMs. The primary mandate of our group is to develop and improve protein characterization and quantitation techniques. Such complexity, both in terms of number of unique biomolecules and variety in chemical composition, requires complexity reduction through the development of separations/enrichment and improvements in mass spectrometric characterization techniques.

Q: Shotgun proteomics is considered to be a rapidly growing field of research, rivalling that of genomics. Do you agree and, if so, why do you think this is?

A: Certainly, shotgun proteomics is the de facto method to characterize proteomes. Advances in mass spectrometers and the required computational tools have also improved dramatically. The current generation of mass spectrometers are capable of sequencing up to 50 peptides per second and can detect over 4 orders of magnitude. A single proteomic experiment will now generate millions of sequencing events. As our understanding of the generated data has improved so have the algorithms. We can now, without human intervention, assign peptide sequences to the mass spectrometric data for the majority of the common peptide classes. These same algorithms can also then assign these peptides to proteins with the help of genomic data. The current state of play is that within a week, we can now identify and quantify over 10 000 proteins. Our laboratory and a few others can perform the same task in a few days primarily as a result of further improvements in chromatography. This level of data is rivalling the level of information generated by genomics yet proteins are the main protagonists in a cell. Thus, we can now study the behaviour of cells in a more biologically relevant manner in the context of their environments and their reactions to stimuli.

Q: Shotgun proteomics could potentially be used in biomarker identification methods in the diagnosis of disease. Do you think this is feasible in a clinical setting? Are there other potential applications?

A: A significant number of diseases and cancers are caused by proteins malfunctioning which has a direct knock-on effect on other proteins. Detecting the malfunctioning of a protein and its direct consequences is useful for both diagnosis and prognosis as well as therapies. A number of these proteins can be shred from tumours into the bloodstream too. The massive improvements observed in proteomics are now allowing efforts to be made to detect such aberrant processes initially in cell lines and tumours but also, ultimately, in blood. Of course, understanding how cellular systems operate (and their impact on physiology) is the greater goal.

Q: You have recently optimized a protocol for the use of Zwitterionic chromatography-cholinate. hydrophilic interaction chromatography (ZIC-cHILIC) for sample separation in proteomics analysis. Can you outline the basic principles of the approach you used and how this is different to currently used protocols?

A: A proteomic experiment usually involves a number of rounds of peptide fractionation followed by LC–MS where the optimal configuration for this final step is the use of nanolitre flow-rate reversed-phase (RP)chromatography and a high sequencing rate mass spectrometer with an electrospray source. The upstream fractionation must be orthogonal to this reversed-phase chromatography. Furthermore, sensitivity of an experiment depends heavily on minimizing surface areas and sample dilution. Developing a high-resolution separation that operates at very low flow rates is desirable. Ion exchange chromatography (IEC) has been the preferred strategy for a decade. It has excellent orthogonality to RP but has a poor separation power for peptides and is difficult to miniaturize. ZIC-cHILIC allowed us to operate with salt-free eluents thus allowing miniaturization, and it also provides an excellent separation that is mostly orthogonal to reversed-phase chromatography.

Q: Single-cell proteome analysis is the ultimate goal for the development of research protocols. How will optimization of sample preparation protocols advance research to this?

A: The ability to analyse material levels approaching single cell populations is the holy grail of most biotechniques. However, most cell behaviour depends on their immediate environment and they are also constantly communicating with their surrounding cells. I would suggest the goal has slightly changed to measuring the proteomes of cell populations at the resolution of a single cell. Nevertheless, the argument still boils down to sample handling, reducing sample losses, improving sensitivity and speeding up the process. Miniaturization and automation will improve sample handling and that requires constant improvement in separation power and robustness. Through improving separations upstream sample manipulation can be reduced. The added bonus of better separations is sharper peaks which lead to less ion suppression in mass spectrometry, improved dynamic range and better sensitivity. Better separation also means faster analysis, which is important because sample throughput is a major challenge too.

Q: Is it plausible that with the information encoded within the proteome of a single cell that disease biomarkers may be identified?

A: I don’t think a single cell is particularly informative; however, catalogues of shred proteins from tumours into the blood stream are being generated in a number of proteomic laboratories. Enzyme-linked immunoassay absorbent (ELISA) has shown that such proteins can be used for prognosis/diagnosis of diseases and, I believe, the current improvements in proteomics will allow the mass spectrometer to replace ELISA with the added benefits of superior specificity, sensitivity and reproducibility.

Q: What are the challenges you face in your field of research?

A: Although great strides are being made in the analytical tools for the characterization of proteomes they are far from comprehensive. Over 200 PTMs exist and methods are only available for a handful. Much work needs to be done before we have a complete picture. An equally significant challenge is stitching all the data together. Bioinformatics is always lagging a little behind what can be experimentally generated, which is primarily a cause of the unknown territory that is being explored.

Q: Is there anything you would like to add?

A: I would add that there is great excitement in the field because we can now identify proteins at a similar depth as genomics and there is now much we can explore about the cell that wasn’t possible in the past. Furthermore, there are still many things to explore in terms of analytical tools to address the trials ahead. Like all scientists, we love a challenge and it’s great because there is a feeling we are up to the task.

Dr Shabaz Mohammed received his PhD specializing in mass spectrometry from the University of Manchester, Manchester, UK, before joining the University of South Denmark as a post-doctoral researcher. He is now an assistant professor at Utrecht University, Utrecht, The Netherlands, and a theme leader at the Netherlands Proteomics Centre. Mohammed’s primary research focus is on the development of proteomics techniques to answer biological questions.

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