Label-free ligand fishing

Surface plasmon resonance (SPR) is an emerging technique in the bio and life-science markets. It offers a new generation of label-free biomolecular analyses, providing information on kinetic processes (association and dissociation), binding affinity, analyte concentration and real-time molecule detection. It has become a powerful tool for the analysis of biomolecular events involved in drug development, cancer research, antibody screening and more.

The phenomenon of SPR occurs when light interacts at the interface between a biochip and a liquid medium. It permits researchers to follow modifications of the refractive index (or the reflectivity) in real time. Such modifications are induced by a biomolecular interaction between immobilised ligands (probe molecules) and captured analytes (target molecules). SPR monitors these changes of reflectivity to characterise the biomolecular events (such as binding and dissociation) occurring at the surface of the biochip in real time.

The SPR imaging (SPRi) technology offered by Horiba Scientific-GenOptics takes SPR analysis a step further. The SPRi-Lab+ and SPRi-Plex II instruments enable visualising the whole biochip surface in real time using a video CCD camera. This design allows biochips to be prepared in an array format; with each spot corresponding to a specific immobilised ligand. Up to several hundreds different molecules can be spotted using an automated spotter, opening the way to high throughput information for biomolecular interaction. The multiplexing capabilities of SPRi can meet with any experimental design without concession. Meanwhile, the sensitivity of SPRi is not compromised, as analyte concentrations can be detected down to the nanomolar range.

The applications of SPRi are vast and include, for example, protein:protein, DNA:DNA, peptide:protein, polysaccharides:protein or protein:cell interactions. The flexibility of the instruments enables complex samples such as serum and plasma to be analysed for clinical applications.

The coupling of SPRi biosensors and matrix-assisted laser desorption ionisation mass spectrometry (MALDI-MS) is an innovative approach for biomarker discovery in biological fluids. It permits captured analytes to be identified by their molecular weight and sequence (ligand fishing). The main clinical challenge now is to find new types of specific biomarkers. SPRi-MS could help quantify and identify proteins of interest, characterise and quantify their post-translational modifications and capture them in biological fluids.

In this context, the complexity lies in the coupling of both techniques. Most strategies require the elution of the bound analyte and its re-deposition on a MALDI plate. This procedure is time consuming and increases the risk for additional cross-contamination risks. The open format of the Horiba Scientific-GenOptics instruments makes MS coupling easier and faster. The SuPRa-MS platform (surface plasmon resonance in arrays coupled with mass spectrometry) combines SPRi and MS in a single biochip. The biochip used for SPRi (SPRi-Slide) is directly transferred to the MS instrument. There is no need to either elute or re-deposit the bound analyte. The MS enzymatic digestion and the deposition of the MALDI matrix are performed directly on the SPRi-Slide. The latter is then directly placed on the MS plate holder.

Proof-of-concept study

A proof-of-concept study of SPRi-MS imaging coupling was performed for the detection of LAG3 recombinant protein, a potential biomarker for breast cancer, in plasma. For this purpose, a mouse antibody (IgG2A, A9H12) directed against LAG3 was immobilised on a SPRi-Slide using a dedicated surface chemistry (named CS). This chemistry allows direct MS analysis on the SPRi-Slide. Before injecting LAG3, rat serum albumin (RSA) was used to avoid non-specific binding on the surface of the biochip. Then, the specific interaction of LAG3 and A9H12 was monitored using SPRi and images of the interaction were studied. Captured LAG3 molecules where then subsequently analysed using a MALDI-MS imager (Ultraflex, Bruker Daltonics). By showing the distribution of MS peaks specific to LAG3 and RSA respectively, it was possible to build the MS image of LAG3 spots directly on the SPRi-Slide.

The SuPRa-MS platform pioneers the combination of SPR imaging and MS imaging (MSi). It offers the possibility to gain spatially resolved information on the capture, sequence and molecular weight of clinical biomarkers.

Multiplexed SPRi analysis provides rapid and high-throughput information in real time from up to several hundreds interactions in parallel. The technology is sensitive and does not require the use of labels. It can speed-up the workflow and reduce consumable costs during optimisation processes. The coupling with MS analysis is straightforward and easier, which makes it a valuable tool for biomarker identification.