Development of online bioaffinity-high resolution mass spectrometry

The online bioaffinity-mass spectrometric approach developed and described in the previous chapter provides the structural characterisation of the eluted peptides or proteins by mass spectrometry. This interface was therefore considered suitable for the development of an online coupling of the SAW biosensor with a high resolution ESI-FTICR mass spectrometer² [168]. A unique attribute of FTICR MS is its capaability to simultaneously provide high mass resolution (>10⁶), and high mass determination accuracy (<1 ppm), along with high sensitivity. The online coupling was developed using a Lysozyme-anti Lysozyme antibody as a model system.

A second interface was developed to provide the advantages of automatic switching of two valves. The new interface was applied to the online coupling of the SAW biosensor with both, ESI-Ion trap and -high resolution-FTICR mass spectrometers. The multi valve-interface used for this online coupling is illustrated in Figure 19. It makes use of two six-port valves and a micro-guard column for desalting and in-situ concentration of protein samples dissociated from the protein-ligand complex³ [169].

2 The online coupling of SAW biosensor with ESI-FT-ICR mass spectrometry developed here has been employed partially by Nicole Engel in her Master thesis [168].

3 The coupling interface developed here has been employed by Alexandru Cozma in his Diploma thesis [169] in the development of a software for the online coupling of SAW biosensor with ESI-FTICR mass spectrometry.

2

4

3 6

5 1 2

4

3 6

5 1

MS Guard column S-Sens K5 Biosensor

waste waste

Pump system

Figure 19: Schematic representation of the two six-port-valve micro-column interface -guard column applied to the online coupling of the SAW biosensor with both, ESI-Ion trap and -high resolution-FTICR mass spectrometers.

The online coupling of a SAW biosensor with a high resolution electrospray-mass spectrometer was performed in three steps as illustrated in Figure 20. The association of the protein(s) was monitoried by the SAW biosensor and was followed by elution of the protein(s) into the guard column at acidic pH conditions (Figure 20a). By connecting the waste capillary of the SAW biosensor to the interface (X valve inject unit, position I), guidance of the eluent was obtained. A 0.3 % aqueous HCOOH solution was used for the removal of buffer salts by switching the X valve inject unit from position I to II so that the buffers are washed out into the waste (Figure 20b).

Elution of the protein(s) from the guard column was achieved using 0.3 % HCOOH/80

% acetonitrile and the transfer of the sample solution into the ESI source was performed by simple switching of the Y valve injector unit to position I (Figure 20c), using a pumping system for elution. In the present interface, connections were made using a standard fused silica capillary with an ID of 153 µm.

2

MS Guard column S-Sens K5 Biosensor

waste waste

MS Guard column S-Sens K5 Biosensor

waste waste

MS Guard column S-Sens K5 Biosensor

waste waste

Figure 20: Schematic representation of the switching positions of the two six–port-valve micro-column interface applied to the online coupling of the SAW biosensor with both, ESI-Ion trap and -high resolution-FTICR mass spectrometers. The waste capillary from the SAW biosensor is connected to the interface in position 1 (the X valve inject unit), the guard column is implemented between the two six valve injector units (X valve, position 2 and Y valve, position 1) and the ESI-MS inlet capillary in position 2 (Y valve). Dissociation of the protein(s) is achieved by eluting with acidic glycine buffer or hydrochloric acid into the guard-column interface (a), followed by cleaning with washing solution (b). The eluate is transferred into the ESI source by switching of the injector and using elution with aqueous acetonitrile/HCOOH as described in the experimental part (c) (Chapter 3.5.7.2).

The efficiency of the multi-valve interface-guard column was demonstrated by varying the flow rates and it was observed that higher flow rates are suited for this approach.

In Table 2, the optimised parameters applied in the online coupling with a high

resolution mass spectrometer (ESI-FTICR-MS), and those used for online coupling with an electrospray ion trap mass spectrometer (ESI-Ion Trap MS) are compared.

Table 2: Conditions for online bioaffinity-mass spectrometric approach.

Electrospray conditions Mass Spectrometer Nebulizer

[psi]

Dry Gas [L min^-1]

Temperature [°C]

Flow rate [µL min^-1]

ESI-FTICR-MS 20 10 150 20-30

ESI-Ion Trap MS 15 9 200 70-80

The Total Ion Chromatogram (TIC) showed to be very important in the online bioaffinity-mass spectrometric approach, due to the polymer contamination of the mass spectrometer during the elution process. Acquisition of MS data via TIC recording eliminates these contaminations from mass spectra. Online bioaffinity-mass spectrometry (Figure 21a), and online bioaffinity-high-resolution mass spectrometry (ESI-FT-ICR-MS) (Figure 21b), respectively, show an elution process of approximately one minute in the MS data acquisition.

0 1 2 3 4 5 6 7 8 autosampler to the multi-valve interface-guard column/C4; (a) TIC-ESI-Ion Trap MS includes also the 10 min wash time of the guard column from salts; (b) TIC-ESI-FTICR-MS contains only the elution of the bound protein from the guard column.

A successful online coupling of the SAW biosensor with the high-resolution FTICR mass spectrometer was obtained using the model system, Lysozyme-anti Lysozyme antibody (Figure 22). The anti-Lysozyme antibody was immobilised on an SAM-COOH (Figure 22a, 2) sensor chip after a pre-activation of the free SAM-COOH-groups by EDC/NHS (Figure 22a, 1) as described in the experimental part. The remaining EDC/NHS-activated groups were capped by ethanolamine (Figure 22a, 3). Lysozyme was flown over the surface to allow binding to the antibody (Figure 22b, 4) and was then eluted from the surface by 0.1 M HCl (Figure 22b, 5). The bound Lysozyme/elution fraction was successfully identified by ESI-FTICR-MS (Figure 22d).

A 5 µl of 1 µM Lysozyme (representing 5 pmol) was characterised by ESI-FTICR-MS before the binding affinity experiment (Figure 22c). TIC trace acquisition during the online SAW-ESI-FTICR-MS proved to be very useful in MS data acquisition, since contamination of the MS spectra was removed in the TIC trace.

0 1000 2000 3000 4000 using online coupling of SAW biosensor with high-resolution ESI-FTICR-MS. The SAW curve illustrates (a) immobilisation of polyclonal anti Lysozyme antibody, followed by (b) Lysozyme binding and elution curves. ESI-FTICR-MS spectra illustrate the bound (c) and SAW eluted (d) Lysozyme.

Lysozyme antibody system was achieved by the online SAW-ESI-FTICR-MS combination. For the kinetic analysis serial dilutions of Lysozyme were injected, and determinations performed by extracting the data from the sensor signals for all concentrations employed. Determinations of dissociation constants were performed by plotting the pseudo first-order kinetic constant (k_obs) versus the Lysozyme concentration and applying linear regression for K_D = k_off* k_on^-1. This resulted in a K_D of approximately 300 nM as shown inFigure 23.

In a separate determination, the obtained K_D value and the K_D value from the reverse systemand the anti-Lysozyme antibody used as the analyte were found comparable (Appendix 3).

0 250 500 750

Figure 23: (a) Association and dissociation curves of Lysozyme with the immobilised polyclonal anti-Lysozyme antibody, and (b) K_D determination of the immune complex. Plotting the K_obs versus used Lysozyme concentrations and appling linear regression provides a KD of approximately 300 nM.

2.2 Application of online bioaffinity-mass spectrometry to the