Project: Absolute Quantification of Proteins in Mass Spectrometers for Diagnostic Use

In this project, we will develop a novel way to quantitatively measure protein concentrations in biofluids. We expect that this technology will be widely applied in medical diagnostics and proteomics research, a multi-billion euro market in which mass spectrometry plays a key role. We aim to valorize this technology by founding a company during the course of this project._x000D__x000D_Proteomics is the study of expressed proteins in a biological system and is important in understanding and combating disease through discovery of disease biomarkers and identifying protein targets in drug discovery. An analytical technique that is often used to quantify and identify peptides and proteins in complex mixtures is liquid chromatography coupled to mass spectrometry, using an electrospray ion source (ESI-LCMS). Proteins are cleaved into short peptides, that are separated by liquid chromatography and subsequently isolated and fragmented in a mass spectrometer. The resulting peptide fragment mass spectra are matched with mass spectral databases to deduce the identity of a protein. Currently widely used in research, it is expected that this method will find its way into routine diagnostics within 5-10 years._x000D__x000D_The quantification of differences between physiological states of a biological system is among the most important and challenging tasks in proteomics. Relative quantification methods dominate the quantitative proteomics field. Relative quantification provides information regarding specific protein abundance changes between two conditions, such as healthy and diseased for instance. Many of the relative quantification methods rely on stable isotope labelling to create mass tags that can be recognized by mass spectrometry and provide the basis for differential quantification. There is an urgent need, however, for absolute quantification. For application in a clinical setting of biomarkers that are discovered by proteomics, it is important to know the absolute amount of a protein and the intra- and interindividual variation of a biomarker. In drug discovery, absolute protein concentrations are valuable to understand the biology guiding the response to a drug._x000D__x000D_A drawback of the ESI-LCMS technique is that it is not inherently quantitative as molecules exhibit a wide range of physicochemical properties such as size, charge, pKa, hydrophobicity etc. This leads to large differences in ionisation and mass spectrometric response. When using calibration curves of appropriate internal standards with similar properties as the molecule of interest, however, this can be circumvented and absolute concentrations can in principle be determined by ESI-LCMS. This was already shown by the Erasmus MC and others (van Kampen et al. 2008) Currently peptides containing stable isotopes are used as internal standards. However, these are very expensive and lack standardization, preventing their broad application. _x000D__x000D_We propose to develop affordable internal peptide standards as an innovative alternative to current methods. Synthetic peptides will be designed with similar properties, yet distinguishable from the peptide of interest in mass spectrometry. As case study, human reference Cerebrospinal fluid (CSF) will be elaborated but all methods and databases will be developed in such a manner that they can easily be extended to other biofluids._x000D__x000D_Neurodegenerative disorders are disorders in which cells of the brain or spinal cord are lost. Examples are Alzheimer's disease, Parkinson's disease and Multiple Sclerosis. Neurodegenerative diseases form an area of great medical need: in Europe 11.9 per 1000 inhabitants have Alzheimer's disease, for example. Still much is unclear about the etiology of neurodegenerative diseases and no good biomarkers nor treatments exist._x000D_CSF circulates within the ventricles of the brain and the subarachnoid space of the central nervous system and is derived in part from extracellular fluid of the brain. CSF can therefore be considered the best compartment for discovery of diagnostic biomarkers for neurodegenerative diseases and biomarkers that predict drug-related exposure and efficacy for those disorders. With the developed method using synthetic peptides, a database will be designed that contains absolute concentrations of peptides and proteins in human reference CSF._x000D__x000D_The ultimate aim of this proposal is to develop a library of synthetic peptides that can be used for absolute quantification on any mass spectrometry platform. To test this database, CSF from Alzheimer and Multiple sclerosis patients will be measured and compared to the absolute concentrations in the database obtained from the reference CSF material. This would eventually show leads for Alzheimer and multiple sclerosis. The synthetic peptides, the library and the database will be suitable for use in clinical chemistry labs for diagnostics of neurodegenerative diseases in general and in pharmaceutical industry for drug discovery of central nervous system disorders.

Acronym PEPSPEX (Reference Number: 4655)
Duration 01/04/2009 - 30/09/2012
Project Topic A product will be developed for determining absolute peptide and protein concentrations in biofluids for diagnostic purposes using a combination of synthetic peptides, mass spectrometric and database technology
Project Results
(after finalisation)
We have developed a new method that can compete with standard isotope labelled reference compounds that can be used for large sets of different protein quantifications in mass spectrometry.
Network Eurostars
Call Eurostars Cut-Off 2

Project partner

Number Name Role Country
3 Pubgene AS Partner Norway
3 Erasmus MC Partner Netherlands
3 Pepscan Presto BV Coordinator Netherlands