2 RESULTS AND DISCUSSION
2.2 Epitope elucidation of Aβ-specific single-chain antibodies
2.2.3 Mass spectrometric characterization of Aβ-peptides
In order to perform the affinity mass spectrometry experiments and to characterize the identified epitope, several Aβ peptides were synthesized by solid phase peptide synthesis (SPPS) using Fmoc/t-Butyl chemistry.
Aβ(1-40) peptide was first synthesized by SPPS using Fmoc chemistry on a semi-automatic peptide synthesizer EPS-221 (Intavis, Germany) on Rink Amide MBHA resin. The C-terminal amino acid of the peptide is attached to the Rink Amide MBHA resin. The resin is insoluble in the solvents used for the peptide synthesis (e.g. DMF), but has the tendency to swell in volume in these solvents. The amino acids used in SPPS are most often Fmoc based temporary α-amino protected. If the amino acid side chain contains a functional group, a “permanent protected group” such as t-butyl (trityl, 4-methyloxytrityl) is used. The permanent protecting group is usually stable during SPPS and is cleaved together with the peptide from the resin in the last step.
The first step in the SPPS was the removal of the N-terminal Fmoc protecting group from the resin using a mixture of 2 % DBU (1,8-diaza-bicyclo[5.4.0]undec-7-ene), 2 % piperidine in DMF (dimethylformamide). The next step was the activation of the carboxyl group of the N-terminal-Fmoc protected amino acid by benzotriazol-1-yl-N-oxy-tris-pyrrolidinophosphonium-hexafluorophosphate (PyBop/NMM) in order to attach the first amino acid to the resin. The resin was washed with DMF after every synthesis cycle to remove all reagents or by-products which were in excess. The deprotection and coupling steps were repeated until the desired peptide was obtained. After the completion of synthesis, the peptides were cleaved from the resin.
The yield of Aβ(1-40) after solid phase peptide synthesis and purification was around 30 - 40%.
Due to the high hydrophobicity of the C-terminal domain resulting in a high propensity to aggregate in solution as well as on-resin during the elongation of the peptide, Aβ(1-40) was further synthesized on a Tenta Gel RRam resin (Rapp polymers). The resin is employed for difficult sequences. Furthermore, Aβ(1-40), Aβ(12-40) and Aβ(17-28) were synthesized manually by Fmoc/t-butyl strategy on Tentagel RRam resin (Table 3). The Fmoc- deprotection was performed using a mixture of 2 % DBU,
2 % piperidine in DMF. The coupling of the C-terminal of the N-α-Fmoc protected amino acid was carried out in a solution containg a mixture of PyBoP and NMM (N-methyl-morpholine) in DMF. The activated amino acid was then added to the resin.
The efficacy of each coupling step was established by different colorimetric tests, such as Kaiser test, Bromophenol blue and chloranil test. The Kaiser test is based on the reaction of ninhydrin with primary amines, the bromophenol blue test can detect all types of amines and the chloranil test is for the detection of secondary amino groups.
Table 3:Aβ peptides synthesized by SPPS using Fmoc/t-butyl strategy.
66.1 3022.5 / 3022.7
VHHQKLVFFAEDVGSNKGAIIGLMV GGVV
Aβ(12-40)
227 1324.6 / 1324.9
LVFFAEDVGSNK Aβ(17-28)
207 4329.9 / 4329.0
DAEFRHDSGYEVHHQKLVFFAEDV GSNKGAIIGLMVGGVV
Aβ(1-40)
∆m (ppm) [M+H]+calc/[M+H]+exp
Sequence Peptide
66.1 3022.5 / 3022.7
VHHQKLVFFAEDVGSNKGAIIGLMV GGVV
Aβ(12-40)
227 1324.6 / 1324.9
LVFFAEDVGSNK Aβ(17-28)
207 4329.9 / 4329.0
DAEFRHDSGYEVHHQKLVFFAEDV GSNKGAIIGLMVGGVV
Aβ(1-40)
∆m (ppm) [M+H]+calc/[M+H]+exp
Sequence Peptide
After the cleavage from the resin, the synthesised peptides were precipitated with t-butylmethyl-ether, washed with diethyl ether and dissolved in 5 % acetic acid prior to freeze-drying.
The molecular weight of crude peptides was determined by mass spectrometry.
Crude peptides were then purified by reverse phase-high performance chromatography (RP-HPLC) on a Vydac C4 column (250×10 mm I.D; Grace Davison). The column is usually employed for the separation of hydrophobic peptides and proteins. A linear gradient elution (0 min 0 % B; 5 min 0 % B; 50 min 90 % B) with solvent A (0.1 % TFA in water) and solvent B (80 % acetonitrile in 0.1 % TFA) has been applied.
For purified peptides, analytical RP-HPLC using a Vydac C4 column (250×4.6 mm I.D.) with 5 µm silica (300 Å pore size) (Hesperia CA) was carried out and the major peak was analyzed by mass spectrometry. The analytical HPLC of Aβ(1-40) indicated a major peak at 31.9 retention time. The fraction was collected, lyophilized
and analyzed by MALDI mass spectrometry. The MALDI-TOF mass spectrum of the
1000 1500 2000 2500 3000 3500 4000 4500 m/z
MWcalc = 4328.8 Da
1000 1500 2000 2500 3000 3500 4000 4500 m/z
MWcalc = 4328.8 Da MWexp = 4328.0 Da
b
2165.1
4329.0
1000 1500 2000 2500 3000 3500 4000 4500 m/z
MWcalc = 4328.8 Da MWexp = 4328.0 Da
b
2165.1
4329.0
Figure 23: a. RP-HPLC and b. the corresponding mass spectrum of Aβ(1-40).
Aβ(17-28) epitope peptide was synthesized by SPPS. The purified Aβ(17-28) peptide was analyzed by analytical HPLC and mass spectrometry. In the MALDI-TOF mass spectrum of the purified Aβ(17-28) a signal at m/z 1324.9, corresponding to [M+H]+ ion could be identified (Figure 24).
0 10 20 30 40
1200 1700 2200 2700 m/z
1324.9
1200 1700 2200 2700 m/z
1324.9
1200 1700 2200 2700 m/z
1324.9
1346.8
1200 1700 2200 2700 m/z
1324.9
1346.8
a b
Figure 24: Analytical RP-HPLC profile and MALDI-TOF mass spectrum of Aβ(17-28).
After epitope identification (Chapter 2.2.2), the following biotinylated amyloid peptides encompassing the identified epitope were synthesized using Fmoc/t-butyl chemistry:
Biotin-G5-Aβ(1-40), Biotin-G5-Aβ(12-40), Biotin-G5-Aβ(1-16) and Biotin-G5-Aβ(17-28), Biotin-G5-Aβ(20-30), Biotin-G5-Aβ(20-37). Alanine mutated Biotin-G5-Aβ(17-28) peptides were also prepared in order to identify the functional epitope. The syntheses
were carried out on a Rink amide MBHA resin using a semi-automated peptide synthesiser. The activation of the carboxyl groups was performed with PyBop/NMM in DMF. At the N-terminal part five glycines were added. After completion of the synthesis, the N-terminus was biotinylated on the resin using 5 equiv of D-(+) Biotin.
After cleavage of the crude peptide from the resin, the products were purified by semi-preparative HPLC. The pure peptides were characterized by analytical RP-HPLC and mass spectrometry (Figure 25).
10 20 30 40 50
1000 2000 3000 4000 5000 m/z
MWcalc = 4837.35 Da
1000 1500 2000 2500 3000 3500 m/z
MWcalc = 1834.87 Da
1000 2000 3000 4000 5000 m/z
MWcalc = 4837.35 Da MWexp = 4837.45 Da
4838.45
1000 2000 3000 4000 5000 m/z
MWcalc = 4837.35 Da
1000 1500 2000 2500 3000 3500 m/z
MWcalc = 1834.87 Da MWexp = 1834.87 Da
1835.87
b2
1000 1500 2000 2500 3000 3500 m/z
MWcalc = 1834.87 Da
1000 1500 2000 2500 3000 3500 m/z
2465.0861
1000 1500 2000 2500 3000 3500 m/z
2465.0861
MWcalc = 2464.07 Da MWexp = 2464.08 Da
1000 1500 2000 2500 3000 3500 m/z
2465.0861
MWcalc = 2464.07 Da MWexp = 2464.08 Da
c1 c2
10 20 30 40 50 0,2
0,3 0,4 0,5 0,6 0,7
Time (min) Absorbance (mAu)
33.8
1000 1500 2000 2500 3000 3500 4000 4500 m/z
MWcalc = 3530.83 Da MWexp = 3530.85 Da
3531.85
d1 d2
10 20 30 40 50
0,2 0,3 0,4 0,5 0,6 0,7
Time (min) Absorbance (mAu)
33.8
1000 1500 2000 2500 3000 3500 4000 4500 m/z
MWcalc = 3530.83 Da MWexp = 3530.85 Da
3531.85
1000 1500 2000 2500 3000 3500 4000 4500 m/z
MWcalc = 3530.83 Da MWexp = 3530.85 Da
3531.85
d1 d2
Figure 25: a1, a2: RP-HPLC and MALDI-FTICR - mass spectrum of biotin-G5 Aβ(1-40); b1, b2: RP-HPLC and MALDI-FTICR mass spectrum of biotin G5 Aβ(17-28); c1, c2: RP-HPLC and mass spectrum of biotin-G5 Aβ(1-16); d1, d2: RP-HPLC and MALDI-FTICR mass spectrum of biotin-G5 Aβ(12-40).