Development of online bioaffinity-electrospray ion trap mass spectrometry

spectrometric approach (Figure 9): (i) direct coupling of the waste capillary of the SAW biosensor with an electrospray mass spectrometer and (ii) online coupling of the SAW biosensor with an electrospray mass spectrometer via an interface used for desalting and sample concentration (Figure 9).

SAW - Biosensor ESI-MS

Kinetics protein-ligand

Structural characterisation protein-ligand

a.

b. SAW - Biosensor ESI-MS

Interface

Figure 9: Strategies used in the development of the online bioaffinity-mass spectrometry approach. (a) Direct coupling of the waste capillary of SAW biosensor with an electrospray mass spectrometer; (b) online coupling of the SAW biosensor with an electrospray mass spectrometer via an interface used for desalting and sample concentration.

Several important aspects were taken into account during the development of the online coupling: (i) the need for physiological pH 7.4 in the analysis of protein-ligand interactions; (ii) suitable flow rate for the ESI-MS analysis; and (iii) buffers containing low salt concentrations which enable good ionisation process.

In the first strategy (Figure 9a) the waste capillary of an S-Sens K5 Biosensor (SAW Instruments GmbH) was connected directly with the inlet capillary of the electrospray mass spectrometer (Bruker Esquire 3000 ion trap mass spectrometer). A peptide-anti-3-nitrotyrosine antibody was used as model system to study the applicability of the direct coupling of the biosensor with an electrospray mass spectrometry. The antibody was covalently immobilised on the SAM-COOH surface after a pre-activation of the free COOH-groups (Figure 10a, 1). This was followed by affinity binding of the synthetic peptide H-GKRLKNY(NO₂)SLPWGA-OH (Figure 10a, 4) and a dissociation step (6) as illustrated in Figure 10b (5).

0 500

Figure 10: Sensogram of (a) Immobilisation of an anti-3-nitrotyrosine antibody and (b) binding affinity of H-GKRLKNY(NO₂)SLPWGA-OH peptide to the antibody. The left sensogram (a) illustrate injections of EDC/NHS (1) for activation of COOH-groups followed by the immobilisation of the anti-3-nitrotyrosine antibody (2), and capping by ethanolamine (3). The right sensogram represents the association curve (4) and elution curve (5) of Tyr-nitrated peptide [H-GKRLKNY(NO₂)SLPWGA-OH]. The elution or total dissociation of the peptide was done by pH shift using a 50 mM Glycine pH 2 as described in Experimental part (Chapter 3.5.7.1). An increasing of the phase signal was observed for association of the PCS peptide (4) and a decreasing by elution (5).

The synthetic peptide (relative amount 1 pmol) bound to the monoclonal anti-3-nitrotyrosine antibody could be identified with very low relative intensity by electrospray mass spectrometer (Bruker Esquire 3000 ion trap mass spectrometer) (Figure 11).

767.90

1534.76

0 200 400 600 800 1000 Intens.

400 600 800 1000 1200 1400 1600 1800 m/z

[M+H]⁺ [M+2H]²⁺

H-GKRLKNY⁴³⁰(NO₂)SLPWGA-OH

Figure 11: Mass spectrum of the dissociated peptide [H-GKRLKNY(NO₂ )SLPWGA-OH] bound to a monoclonal anti-3-nitrotyrosine antibody. Coupling was achieved by connecting the waste capillary of SAW biosensor directly with the capillary for direct infusion into the electrospray mass spectrometer (Esquire 3000 ion trap mass spectrometer).

The relatively low flow rate, 20-40 µL min^-1, of the SAW biosensor leads to significant dilution of the eluted peptide solution, if no pre-concentration of the sample solution for mass spectrometric analysis was performed. Also, the buffer containing high salts concentrations represents a significant disadvantage for the ionisation of the eluted protein solution in the mass spectrometer. Therefore, an interface was developed and applied in a second strategy (Figure 12). The interface developed for online coupling of SAW biosensor with an ESI-Ion trap mass spectrometer utilises a six-port valve micro-column and micro-injector for desalting and in-situ concentration of protein samples dissociated from the protein-ligand complex [141, 167] as illustrated in Figure 12¹.

1 The interface developed herein has been used for interactions studies of amyloid beta and synuclein by Stefan Slamnoiu in his diploma thesis [167].

2

6

1 4

5

3 _{5 µm frit}

1.5 cm x 1 mm packed 40 µm particle size 5 µm frit

1.5 cm x 1 mm packed 40 µm particle size

Guard column

a b

Figure 12: Schematic representation of the six-port-valve micro-column interface (a) and of the guard column (b) used for desalting and in-situ concentration of protein samples dissociated from the protein-ligand complex [141].

The online coupling of SAW biosensor with an electrospray ion trap mass spectrometer was performed manually via two flow systems (Figure 13). After SAW detection of the protein-ligand association and elution of the protein(s) into the guard column using a by pH shift (Figure 13a, flow 1), the protein(s) sample was washed with 0.3 % aqueous HCOOH to remove buffer salts. Elution of the protein(s) from the guard column with aqueous 0.3 % HCOOH/80 % acetonitrile into a manual microvalve-injector transferred the sample into the ESI source (Figure 13b, flow 2). In this mode the online coupling was carried out manually by switching the multi valve injection unit. An HPLC-pumping system was used for performing the elution. The interface connections were made using a standard fused silica capillary (ID = 153 µm).

This interface system provided routine mass spectrometric analysis of real time protein association from the biosensor surface, and showed long term operation stability with minimal contamination of the ESI-MS system. [141].

a

500 600 700 800 900 1000 1100 1200 1300 m/z

19+

500 600 700 800 900 1000 1100 1200 1300 m/z

19+

Figure 13: Schematic representation of online SAW-ESI-MS combination of an SAW Biosensor instrument with ESI-MS using a six-port-valve micro-column interface. The waste capillary from the SAW Biosensor is connected to the interface in position 6 and the ESI-MS inlet capillary in position 3. Total dissociation of protein(s) from the antibody surface was performed by elution with acidic buffer to the micro-column interface (flow 1). This was followed by cleaning with washing solution, elution with aqueous acetonitrile/ HCOOH , and transfer of the eluate into the ESI source by switching of the injector as described in the experimental part (flow 2) [141].

The sensitivity of the ion trap mass spectrometer using the valve interface-guard column system was evaluated by repeated mass spectrometric analysis of Lysozyme protein and peptide [H-GKRLKNY(NO₂)SLPWGA-OH] (Figure 16, Figure 18).

Therefore, the peptide or protein respectively, was repeated times injected into the micro-column interface and the elution fraction was in dublicate analysed by ESI-MS.

The relative intensity of the most abundant ions of the synthetic peptide H-GKRLKNY(NO₂)SLPWGA-OH, the protonated doubly charged ion, m/z 768.1 was used for quantitative estimation of the sensitivity of the ESI-mass spectrometer (Figure 14).

Figure 14: ESI-MS spectrum of 82 pmol H-GKRLKNY(NO₂)SLPWGA-OH peptide injected into the valve interface-guard column. Singly and doubly charged ions were identified.

The relative intensity of m/z 768.1 was determined from duplicate experiments and averaged at all peptide concentrations used. These results are illustrated in Figure 16.

The data shows that ca. 0.1 pmol bound to the valve interface-C18 guard column could be still analysed in the ESI-mass spectrometer, due to the sample concentration and desalting via the six-valve-guard column interface (Figure 15).

513.2

Figure 15: Comparison of the ESI-ion trap mass spectra of (a) 0.1 pmol and (b) 16.3 pmol synthetic peptide H-GKRLKNY(NO₂)SLPWGA-OH. (a) The mass spectrum provides a very low intensity peak which is in the lower limit of detection.

0,0E+00 5,0E+06 1,0E+07 1,5E+07 2,0E+07 2,5E+07 3,0E+07 3,5E+07

0,081 1,630 325,945 6518,905 16297,262

n (pmol)

Relative Intensity

0,081 0,163 0,815 1,630 16,297 81,486

0,0E+00 1,0E+05 2,0E+05 3,0E+05 4,0E+05 5,0E+05

H-

⁴²⁴

GKRLKNY

⁴³⁰

(NO

₂

)SLPWGA

⁴³⁶

-OH

Figure 16: Sensitivity of the ESI-Ion Trap MS response by injection of different concentrations of synthetic peptide H-GKRLKNY(NO₂)SLPWGA-OH.

The ionisation of the peptide depends on the peptide structure and the solvent accessibility to the protonated macromolecule. Different amount of peptide was immobilised on the guard column and injected via interface to the ESI-Ion Trap mass spectrometer (Bruker Esquire3000plus) and till 0.1 pmol peptide could be detected by ESI-mass spectrometer.

The complexity of protein conformations may affect the ability to ionise and therefore, lead to variable results in ESI-MS. For the relative sensitivity measurements, Lysozyme, a 14.3 kDa protein was used and the mass spectrometric structural characterisation performed by directly coupling of a C4-guard column to the mass spectrometer (Figure 17). Relative intensities of m/z 1590.5 from duplicate experiments were averaged for all protein concentrations and the data are presented in Figure 18.

1301.6

800 1000 1200 1400 1600 1800 2000 2200 2400 m/z

[M+9H]⁹⁺

800 1000 1200 1400 1600 1800 2000 2200 2400 m/z

1301.6

800 1000 1200 1400 1600 1800 2000 2200 2400 m/z

[M+9H]⁹⁺

[M+10H]¹⁰⁺

[M+10H]¹¹⁺ [M+8H]⁸⁺

Figure 17: ESI-MS spectrum of 700 pmol Lysozyme injected in the valve interface-guard column. Protonated multiply charged ion formation could be identified.

0,699 3,496 13,982 34,955 69,911 279,642 419,463 559,284 699,105

n

_Lysozyme

(pmol)

Figure 18: Relative sensitivity of the ESI-MS by injection of different concentrations of Lysoyzme into the valve interface-guard column. The ionisation depends on the possibility of ionisation of the macromolecules based on the structure and accessibility of the solvents to protonated the macromolecule. The relative sensitivity is shown for 14.3 kDa Lysozyme used in the online coupling.

Im Dokument Development and bioanalytical application of affinity-mass spectrometry for identification and structural characterisation of protein-ligand interactions (Seite 37-46)