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While completion of gene and protein sequence database has been relatively straightforward, the functionality of proteins, being a result of many mutual interactions, is difficult to discover and define. This is because interactions of over 100.000 proteins contained in a cell cannot be deduced from their identification alone.

The effective strategy for prediction of functional properties or drug-ligand interactions will use as the first step study of the binding of target molecules to selective ligands immobilized on a surface, followed by their elution into electro spray mass spectrometer ( ES-MS), for their identification and quantification. Presently, in order to get an overview of proteins expressed at one time, 2D gel electrophoresis is combined with MS, yet this approach does not offer a clue as to protein functionality or the binding /dissociation of the drug/protein complex.

Affinity binding, on the other hand, is related to functionality of interacting species and will separate targeted biomolecules from the protein pool. The accurate molecular mass determination of the separated biomolecules will be determined by ES-MS – a simplified task as the mass spectra will be less complex since irrelevant matrix has been removed. This is why recently a merger of biomolecular interaction analysis (BIA) with ES-MS has been described, whereby BIA serves as a selective capture system.

This innovative approach has the following advantages:

BIA allows real time kinetic analysis of interacting biomolecules

BIA can be carried out using a variety of immobilized ligands BIA used microfluidic manipulations that facilitate selective elution into ES-MS using appropriate modifiers, while unwanted matrix is diverted to waste.

BIA can be carried out in two different ways: either on a thin flat substrate (“chip”) that is probed by surface plasmon resonance (SPR) or on a renewable microcolumn, packed by microbeads and probed by UV-VIS spectrophotometry or by fluorescence.
(see Bead Injection) . There are several advantages to Bead Injection BIA:

It provides spectral resolution that assists in identification of target molecules during their capture and elution

The microcolumn has much larger surface – and binding capacity than a flat surface of a microchip

Beads need not to be regenerated –as they can be automatically replaced by microfluidic manipulation

Flexibility: since the beads used are identical with supports for affinity chromatography, the re commercially available with a broad variety of immobilized functional groups (avidin, streptavidin, protein A, protein G, etc)

Microlumns can be eluted or beads with captured target molecules can be recovered or reused.

Low cost

Both SPR-BIA / 1 / and BI-BIA / 2 / have been successfully coupled with ES-MS. Since all components of the system are commercially available, the merger of these sophisticated techniques is no longer only a drawing board item, but a real tool for for proteomics, immunoassays, diagnostics and drug discovery.

References

1/ “ BIAcore and Mass Spectrometry: More than Just a Marriage of Convenience”
C.P. Sonksen,  BIAjournal, 7 ( 2001) , 19., also C.P. Sonksen e.t al  Anal. Chem 70 ( 1998) 2731.