A new online bioaffinity-electrospray ionisation (ESI) mass spectrometry approach enables the simultaneous label-free detection, identification and quantification of protein-ligand interactions.
The approach combines the sam5 surface acoustic wave (SAW) biosensor from Saw Instruments directly with ESI-MS and enables the direct connection of protein-ligand KD analysis with their subsequent quantification and structural characterisation by mass spec.
This tandem Saw-ESI-MS system has been developed in the laboratory of Prof Michael Przybylski at the University of Konstanz and recently published (Dragusanu et al, 2010)1.
Biosensors have previously only ever been connected to mass spec indirectly in a MALDI-TOF format where crystallised sample spots are analysed.
The new system is the first that directly connects liquid flow from the Saw biosensor directly into the ESI-MS via a standard desalting interface.
This novel set-up enables direct quantitative determinations of protein-ligand complexes by Saw-yielded dissociation constants (KD) from low-nanomolar to sub-micromolar sample concentrations.
The Przybylski team observed the key advantages of Saw in comparison to classical immuno-analytical bioaffinity techniques to be the direct and rapid determination of association/dissociation constants from small and dilute sample amounts, without the need for labelling or recalibration for buffer changes.
Furthermore, the sam5 biosensor is able to make detection measurements in complex biological samples - for example, crude brain samples, blood or cell lysates.
The Saw-ESI-MS technique has been successfully applied by the Przybylski group to a number of clinically relevant applications for biomarker discovery, analysis and epitope mapping.
These include human B-amyloid peptides (Alzheimer's Disease), Substance P peptide-calmodulin complex, tyrosine-nitrated peptides (neurotoxic events) and human B-synuclein (Parkinson's Disease).
No details on affinity binding and KD determination of the anti-a-synuclein-human a-synuclein complex have previously been reported.
'We are confident that the new online Saw-ESI-MS approach utilising the sam5 biosensor will develop into an efficient and sensitive tool for the label-free detection, identification and quantification of biopolymer-ligand interactions as diverse as antigen-antibody and lectin-carbohydrate complexes,' said Prof Przybylski.
'Certainly SAW technology is now becoming increasingly popular for the study of biomacromolecular interactions due to its high detection sensitivity in dilute solutions and the results that we have obtained to date suggest a broad range of potential applications for this exciting new technique,' he added.
The sam5 biosensor instrument enables advanced real-time biomolecular interaction and kinetic studies.
The sam5 is a peerless biosensor utilising Surface Acoustic Wave (SAW) technology for the label-free detection of real-time binding and structural events.
Dr Thomas Gronewold from Saw Instruments will present at the Affinity-Mass Spectrometry Workshop on 18-19 November 2010 at the University of Konstanz, Germany.
