Getting to the heart of the problem

Hailed as the next step in biological research, proteomics could provide answers in many fields. But can it be combined with stem cell research to give solutions for cardiovascular research? 

PROTEOMICS is the large-scale analysis of proteins and is considered the next step in the study of biological systems. Although stem cell therapy for regenerative medicine and tissue engineering has great potential, the mechanisms of how stem cells differentiate to cells of the cardiovascular lineage are still unclear. Much previous research has been focused on gene expression, but proteomics is capable of advancing the understanding of stem cell differentiation beyond the genetic level by interrogating protein modifications.

The emergence of advanced proteomics solutions allows researchers to uncover new insights into stem cell differentiation, which could not have been obtained by conventional techniques. The application of this approach could lead to new strategies for treatments and possible cures for cardiovascular conditions.

The latest advances in proteomics are being applied by The Vascular Proteomics Group at the James Black Centre, King’s College London. This facility has a range of core competencies including genomics, proteomics, multiphoton confocal microscopy and MRI. In December 2007, the Cardiovascular Division was awarded a £9m Research Excellence Award from the British Heart Foundation and a portion of the grant will be used to facilitate the development of proteomic research into cardiovascular disease. One main focus of King’s College is to establish how stem cells can repair vessels or ischemic heart tissue.

The studies undertaken by the Vascular Proteomics Group aim to decipher the distinguishing proteomic and metabolic features of stem cell-derived cardiovascular cells. The overall aim is to identify key proteins or small molecules that may be drug targets for promoting stem cell differentiation, as stem cell research holds great promise for regenerative medicine and tissue engineering.

Previous studies have been limited to characterising stem cells based on the expression of surface markers. However, the presence or absence of cell surface markers does not necessarily tell much about the activation state of the cell. This, however, could explain why stem cells injected into one patient are more potent than in another patient.

The group aims to develop a comprehensive picture of the nature of the cells that are being injected into patients and their molecules. This is achieved by isolating the stem cells and analysing their secreted factors. While standard ELISAs can look at molecules one at a time, mass spectrometry allows a comprehensive detection of all proteins above a certain threshold. The complexity of the secretome is quite limited, thus, even proteins present at the levels of nanograms per millilitre, such as cytokines and chemokines, can be identified.

As the research continues, King’s College will isolate stem cells from patients, characterise the secreted factors and try to establish how stem cells deliver benefits to clinical outcome.
 
The sensitivity and robustness of the instruments was essential to the studies

'Cell culture flask' courtesy of the British Heart Foundation

being undertaken by the Vascular Proteomics Group. The researchers and technicians at King’s College are biomedical researchers and therefore, the group needed a highly reliable solution that was user-friendly, requiring little day to day maintenance and delivering highly accurate data. A solution that allows the sequencing of peptides and provides unequivocal protein identification was a necessity. King’s College requires cutting-edge technology to complement the research centre’s multidisciplinary environment and core facilities.

As a result, the centre looked for a system that would be sufficiently fast and powerful yet require minimal maintenance in order to develop a high throughput method for the identification of complex proteins. This would allow the team to accurately analyse many samples. The instrumentation also needed to be highly sensitive and reliable in order to use proteomics to ensure that cells are differentiated and are safe to be used, either for grafts or for tissue repair.

For this specific research, King’s College required a highly sensitive instrumentation setup that could process a high number of samples and analyse fairly large molecules at low levels of concentration.

King’s College purchased the combination of a high performance mass spectrometer, protein identification and biomarker discovery platform (Thermo Scientific LTQ Orbitrap XL), along with a linear ion trap (Thermo Scientific LTQ XL) coupled with electron transfer dissociation (ETD) capability. These instruments provide the high quality and sensitivity that was essential for the research.

Using a high performance linear ion trap enables King’s College to deliver more structural information than traditional ion trap mass spectrometers and the ETD option provides sequence information not available from conventional analysis methods. The research group has found that by rapidly alternating different fragmentation techniques, it is possible to significantly increase proteome coverage and have greater confidence in protein modifications identified. The Vascular Proteomics Group believe that ETD technology is the future in proteomic research and therefore want to be among the first to use such cutting-edge equipment. ETD offers the most forward-looking technology in protein analysis and in the future, the group believes that ETD will be a widely applied fragmentation technique in proteomic research.

The high mass accuracy and resolution of the Orbitrap XL allows King’s College to investigate the secreted factors of different stem cells. It is possible to achieve a reliable match even for low abundant proteins and the accurate mass adds confidence for the analysis of peptides in a complex mixture.

The combination of the different instruments provides a comprehensive solution for complex protein analysis and the intelligent sequencing of peptides.

The Vascular Proteomics Group utilises proteomics in stem cell and cardiovascular research. The potential implications of this research are in terms of clinical therapy. In the long term, the group wants to identify factors, which mediate the benefits of stem cell therapy. From a pharmacological point of view, it would be much better to deliver proteins or small molecules, rather than cells to patients.

The superior expertise offered by the use of proteomics in cardiovascular research will help today’s researchers to advance their understanding of cardiovascular diseases and contribute to new drug discovery and stem cell-based therapy in the future.