A "Shotgun" Approach to Proteomics

Dr Shabaz Mohammed from Utrecht University, The Netherlands, spoke to LCGC Europe 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.

To view the extended interview, watch out for the special proteomics issue of

The Column

in March 2013.

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.

E-mail: S.Mohammed@uu.nl

Website: ref=http://bioms.chem.uu.nl>http://bioms.chem.uu.