Figure 5.11 illustrates the basic performance of the on-line MS assay. For comparison, a homogenous fluorescence assay has been set up in parallel. For this purpose, the carrier flow was split after the second microcoil reactor, with 90% of the total flow being directed to a fluorescence detector (Fig. 5.11a) and 10% to the MS (Fig. 5.11b). The affinity interaction between streptavidin and biotin was chosen to study the characteristics of an on-line MS biochemical assay. Fluorescein-biotin was used as reporter ligand for both fluorescence and MS in the SIM mode (m/z 390) detection. In the fluorescence mode, the homogeneous biochemical assay is based on the quenching of the fluorescein-biotin fluorescence upon binding to streptavidin.
At point (1) in Fig. 5.11a, solely carrier buffer is pumped by all pumps (carrier pump, affinity protein pump, reporter ligand pump) resulting in stable baseline in both detectors. At point (2), fluorescein-biotin is added to the reporter ligand pump leading to an increase of the background signal in both detectors. After stabilization of the system, streptavidin is added to the affinity protein pump at point (3). The reaction of streptavidin and fluorescein-biotin leads to an almost complete disappearance of free fluorescein-biotin and, consequently, to a reduction of the baseline to the original level. When injecting active analytes such as biotin (points labeled 4), the concentration of free, unbound streptavidin is reduced in reaction 1, leading to an increase of the free fluorescein-biotin concentration after reaction 2 and a positive signal in both the MS and fluorescence detector. MS is shown to mimic the similar response patterns in the continuous-flow experiment as those observed with fluorescence detection. The decrease of
Fig. 5.11 On-line continuous-flow monitoring of biochemical interaction with (a) fluorescence and (b) MS SIM (m/z 390) detection. Fluorescein-biotin (96 nM), streptavidin (32 nM), 20-mL loop injections of 1000 nM biotin (n = 3). MS instrument: Q-ToF2 (Waters) equipped with a Waters Z-spray electrospray (ESI) source. Point 1: Carrier pump, protein and reporter ligand pumps
Fig. 5.11 On-line continuous-flow monitoring of biochemical interaction with (a) fluorescence and (b) MS SIM (m/z 390) detection. Fluorescein-biotin (96 nM), streptavidin (32 nM), 20-mL loop injections of 1000 nM biotin (n = 3). MS instrument: Q-ToF2 (Waters) equipped with a Waters Z-spray electrospray (ESI) source. Point 1: Carrier pump, protein and reporter ligand pumps are delivering background buffer. Point 2: Fluorescein-biotin (reporter molecule) is added, resulting in an increase of both the fluorescence and MS-SIM signal. Point 3: streptavidin is added, resulting in a decrease of the free fluorescein-biotin concentration. Point 4: injection of the active ligand biotin leads to positive peak due to the displacement of bound reporter ligand.
the unbound fluorescein-biotin concentration upon addition of streptavidin at point (3) indicates that complex formation occurs and that the fluorescein biotin-streptavidin complex does not dissociate during the ionization phase. Complete protein-ligand complexes have been reported to stay intact in the ESI-MS process; however, gentle experimental conditions should be applied.
Furthermore, when using 96 nM fluorescein-biotin and 32 nM streptavidin, an injection of 1 mmol L-1 of biotin results in an almost complete blocking of streptavidin and, consequently, the maximum peak height possible under the current conditions is about 95% of the highest point (3), indicating that apparent binding of biotin to streptavidin is in the order of >95%.
Because the interaction between biotin and streptavidin is strong (Ka = 0.6 x 1015 L mol-1) with a relatively fast association rate (k+1 = 2.4 x 107 L mol-1 s-1) and slow dissociation rate (k-1 = 0.4 x 10-7 s-1), the reaction times are fast, i.e. 10-20 s. Furthermore, the addition of reporter ligand is performed only after the analyte protein reaction has taken place in coil I, avoiding a displacement reaction that would substantially increase the overall reaction time.
Hence the reaction coil volumes were kept as small as possible to reduce band-broadening, i.e. 17 mL and 33 mL, for coil I and coil II, respectively.
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