Project: Find The Needle In The Haystack instrumentation for Mass Spectrometry

The project aims to develop and commercialize an analytical platform for high resolution protein separation and for biomarker discovery. Highly sensitive separation of proteins is a major challenge in biomarker discovery because most chromatographic separation procedures suffer from substantial loss of sample due to unspecific binding to chromatographic materials. In the microfluidic device developed over the past few years there are no solid phase and all separations are performed in the liquid phase. Therefore considerably improved sensitivity is achievable combined with the high resolution obtained in the isoelectric focusing in the device. Therefore the perspectives for observation of low abundant protein and large polypeptide biomarkers in body fluids are improved. _x000D_The consortium plans to use a unique proprietary technology for biomarker discovery in human blood plasma with special focus in Alzheimer disease. Preliminary R&D done by the present consortium is twofold. First, it has been shown that abundant biomarkers are best detected by analyzing a particular isoelectric point (pI) fraction of plasma proteins. These biomarkers are abnormal forms of common proteins rather than totally novel proteins. The abnormality, such as sequence truncation, mutations and posttranslational modifications, often changes the isoelectric point (pI value), and can shift it to the region around pI 7.4, which is avoided by normal protein forms. Thus, disease-related abnormal forms of proteins can be identified under this particular pI window where normal proteins are much less abundant. Second, validated data shows that the proprietary technology owned by the consortium can be used to isolate proteins having a particular pI value and/or separate proteins according to their pI._x000D_The analytical platform utilizes a microfluidic technology which immobilizes molecules in a flow stream according to their electrophoretic characteristics. Since the electrophoretic characteristics of polypeptides are highly dependent on the pH and pI (as well as size), the capture conditions can be tuned to immobilize a particular set of molecules with similar electrophoretic characteristics. Validated data shows that the electrophoretic characteristics of captured polypeptides are highly correlated to their isoelectric point, thus the capture conditions can be optimized to enrich plasma proteins having particular isolectric point value. The novel analytical platform consists of a unique "Findtheneedleinthehaystack"- instrumentation of on-line pI separation of blood proteins with retention of molecules with pI around 7.4, their subsequent on-column digestion and quantitative analysis using LC/MS/MS. This tool, once developed, will allow fast (˜1 h) and clinics-compatible screening of patients for Alzheimer’s Disease biomarkers._x000D__x000D_The other initiative is to use the novel instrumentation as a generic tool for high resolution and high sensitivity on-line pI selection of specific proteins/peptides from complex biological samples and concentrate prior to Mass Spectrometric detection/quantification._x000D_Biomotif has knowledge, IPR and experience in H/D Exchange MS and permeable membrane assisted technology that is a key component in the project._x000D_Karolinska Institutet (Roman Zubarev) has vast knowledge and experience Mass Spectrometry and biomarker research._x000D_SDU (Peter Roepsdorff) has vast knowledge and experience in protein chemistry as well as a world renowned expert in MALDI and ESI Mass Spectrometry._x000D_MSVision has extended knowledge, IPR and long experience in the field of Chromatography & Mass Specrometry._x000D_Biomotif, MSVision, SDU and KI has complementary skills that are needed for the project.

Acronym	NeedleInTheHaystack (Reference Number: 5936)
Duration	01/03/2011 - 28/02/2013
Project Topic	We plan to build a new analytical tool for detecting biomarkers of Alzheimer’s and other neurodegenerative diseases in human blood plasma . _x000D_ This new analytical tool will also provide On-line high resolution pI separation of proteins.
Project Results (after finalisation)	We have developed a "bread-board" prototype pI-Fractionator instrument for mass spectrometry that can help identify more proteins, faster for deep-proteomics applications
Network	Eurostars
Call	Eurostars Cut-Off 5

Project partner

Number	Name	Role	Country
4	Biomotif AB	Coordinator	Sweden
4	Karolinska Institutet	Partner	Sweden
4	MSVision Aps	Partner	Denmark
4	University of Southern Denmark	Partner	Denmark