Supporting Information
Quinacrine reactivity with prion proteins and prion derived peptides
Zbigniew Zawada,1,2 Martin Šafařík,1 Eva Dvořáková,3 Olga Janoušková,3 Anna Březinová,1 Ivan Stibor,1 Karel Holada,3 Petr Bouř,1 Jan Hlaváček,1* and Jaroslav Šebestík1*
Content
- Sequences of the full-length unlabeled (2g) and labeled (4g) prion proteins
- Assignment of NMR spectra
- MALDI of the trypsin cleavage fragments 6a-6e
- ESI and its deconvolution of the full-length unlabeled (2g) and labeled (4g) prion proteins
1. Sequences of the full-length unlabeled (2g) and labeled (4g) prion proteins
HisTag-MoPrP23-230 (2g)
H-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met-Lys-Lys-Arg-Pro-Lys-Pro-Gly-Gly-Trp-Asn-Thr-Gly-Gly-Ser-Arg-Tyr-Pro-Gly-Gln-Gly-Ser-Pro-Gly-Gly-Asn-Arg-Tyr-Pro-Pro-Gln-Gly-Gly-Thr-Trp-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-Gly-Gln-Pro-His-Gly-Gly-Ser-Trp-Gly-Gln-Pro-His-Gly-Gly-Ser-Trp-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-Gly-Gln-Gly-Gly-Gly-Thr-His-Asn-Gln-Trp-Asn-Lys-Pro-Ser-Lys-Pro-Lys-Thr-Asn-Leu-Lys-His-Val-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-Arg-Pro-Met-Ile-His-Phe-Gly-Asn-Asp-Trp-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met-Tyr-Arg-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Asp-Cys-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-Thr-Asp-Val-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-Cys-Val-Thr-Gln-Tyr-Gln-Lys-Glu-Ser-Gln-Ala-Tyr-Tyr-Asp-Gly-Arg-Arg-Ser-OH
HisTag-[Cys(Qui)178,213]MoPrP23-230 (4g)
H-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met-Lys-Lys-Arg-Pro-Lys-Pro-Gly-Gly-Trp-Asn-Thr-Gly-Gly-Ser-Arg-Tyr-Pro-Gly-Gln-Gly-Ser-Pro-Gly-Gly-Asn-Arg-Tyr-Pro-Pro-Gln-Gly-Gly-Thr-Trp-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-Gly-Gln-Pro-His-Gly-Gly-Ser-Trp-Gly-Gln-Pro-His-Gly-Gly-Ser-Trp-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-Gly-Gln-Gly-Gly-Gly-Thr-His-Asn-Gln-Trp-Asn-Lys-Pro-Ser-Lys-Pro-Lys-Thr-Asn-Leu-Lys-His-Val-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-Arg-Pro-Met-Ile-His-Phe-Gly-Asn-Asp-Trp-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met-Tyr-Arg-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Asp-Cys(Qui)-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-Thr-Asp-Val-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-Cys(Qui)-Val-Thr-Gln-Tyr-Gln-Lys-Glu-Ser-Gln-Ala-Tyr-Tyr-Asp-Gly-Arg-Arg-Ser-OH
2. Assignment of NMR spectra
The 1H and 13C chemical shifts were assigned using 2D COSY, HSQC and HMBC data. The strong HMBC correlation between the hydrogens (δ 3.11, 3.35) and carbon (δ 139.3), and between hydrogens (3.14, 3.36) and carbon (δ 139.5), indicated that an acridine moiety was attached to the sulfur of [Cys(Qui)178]MoPrP177 180 (3a) and [Cys(Qui)178]MoPrP176 180 (3b), respectively. A similar correlation between the hydrogens (δ 3.17, 3.28) and carbon (δ 139.3) confirmed the attachment of an acridine moiety to the Cys in [Cys(Qui)213, Asn216]MoPrP210 216 (3e). In the glutathione derivative 3g, a similar correlation between the hydrogens (δ 3.18 and 3.33) and carbon (δ 139.4) also provided an evidence for attachment of an acridine moiety to the sulfur atom.
The assignment of 13C NMR (125 MHz, DMSO-d6) shifts of 3a
The assignment of 1H NMR (500 MHz, DMSO-d6) shifts of 3a
The assignment of 13C NMR (125 MHz, DMSO-d6) shifts of 3b
The assignment of 1H NMR (500 MHz, DMSO-d6) shifts of 3b
The assignment of 13C NMR (125 MHz, DMSO-d6) shifts of 3e
The assignment of 1H NMR (500 MHz, DMSO-d6) shifts of 3e
The assignment of 13C NMR (125 MHz, DMSO-d6) shifts of 3g
The assignment of 1H NMR (500 MHz, DMSO-d6) shifts of 3g
The assignment of 1H NMR (400 MHz, DMSO-d6) shifts of the acridone by-product
The assignment of 13C NMR (125 MHz, DMSO-d6) shifts of the acridone by-product
The assignment of 1H NMR (400 MHz, DMSO-d6) shifts of the thioacridone by-product
The assignment of 13C NMR (125 MHz, DMSO-d6) shifts of the thioacridone by-product
3. MALDI of trypsin cleavage fragments
We used Milli-Q water instead of buffer to prepare all solutions, in order to lower trypsin efficiency, so we were able to obtain long fragments of trypsin cleavage. When buffer was used, we obtained too complicated mixture of short fragments.
Unlabeled (2g) and labeled (4g) prion proteins were cleaved with trypsin: 1 µL of protein solution (2 µg/µL) and 0.5 µL of solution of trypsin solution (1 µg/µL) were added to 18.5 µL of water, and the reaction mixtures were stirred at 38° C for 8 h. The cleaved fragments were then analyzed by MALDI-MS spectrometer.
MALDI of the trypsin cleavage product 6a
Cleaved labeled (black) and unlabeled proteins (blue)
(6a) H-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Asp-Cys(Qui)-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-OH
For C214H310N57O64SCl (mono 4769.22; avg 4772.62) found MALDI-MS, m/z: 4772.0 (M+). The closest fragments in unlabeled cleaved protein: 4769.38 (C210H320N62O60S3), could be present in both labeled and unlabeled protein; not observed in either, and 4860.25 (C205H313N79O59S), could be present in both labeled and unlabeled protein; not observed in either.
MALDI of the trypsin cleavage product 6b and 6c
Cleaved labeled (black) and unlabeled proteins (blue)
(6b) H-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Asp-Cys(Qui)-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-Thr-Asp-Val-Lys-OH
For C262H382N69O83SCl (mono 5889.72; avg 5893.78) found MALDI-MS, m/z: 5893.3 (M+). The closest fragments in unlabeled cleaved protein: 5 875.17 (C263H355N85O73) could be present in both labeled and unlabeled protein; observed in both, and 6 199.80 (C269H411N75O88S3) could be present just in unlabeled protein; not observed.
(6c) H-Glu-Asn-Met-Tyr-Arg-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Asp-Cys(Qui)-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-Thr-Asp-Val-Lys-OH
For C291H425N78O92S2Cl (mono 6583.01; avg 6587.55) found MALDI-MS, m/z: 6588.0 (M+). The closest fragments in unlabeled cleaved protein: 6345.88 (C277H417N77O91S2) could be present just in unlabeled protein; it is unlabeled equivalent of labeled fragment 6c; was observed, and 6828.42 (C301H447N83O96S2) could be present just in unlabeled protein; not observed.
MALDI of the trypsin cleavage product 6d
Cleaved labeled (black) and unlabeled proteins (blue)
(6d) H-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Asp-Cys(Qui)-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-Thr-Asp-Val-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-Cys(Qui)-Val-Thr-Gln-Tyr-Gln-Lys-OH
For C359H527N93O109S5Cl2 (mono 8114.65; avg 8120.84) found MALDI-MS, m/z: 8119.3 (M+). The closest fragments in unlabeled cleaved protein: 7637.49 (C331H511N91O107S5 could be present just in unlabeled protein; was observed, and 8297.75 (C366H511N123O104) could be present in both labeled and unlabeled protein; observed just in unlabeled one.
MALDI of the trypsin cleavage product 6e
Cleaved labeled (black) and unlabeled proteins (blue)
(6e) H-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Asp-Cys(Qui)-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-Thr-Asp-Val-Lys-Met-Met-Glu-Arg-Val-Val-Glu-Gln-Met-Cys(Qui)-Val-Thr-Gln-Tyr-Gln-Lys-Glu-Ser-Gln-Ala-Tyr-Tyr-Asp-Gly-Arg-OH
For C405H590N106O126S5Cl2 (mono 9184.10; avf 9190.91) found MALDI-MS, m/z: 9190.7 (M+). The closest fragments in unlabeled cleaved protein: 8977.99 (C397H592N114O116S5) could be present just in unlabeled protein; was observed, and 9401.34 (C406H617N113O133S6) could be present just in unlabeled protein; was not observed.
4. ESI and its deconvolution of the full-length unlabeled (2g) and labeled (4g) prion proteins
Due to high inaccuracy of MALDI in a higher m/z we performed ESI of the full labeled and unlabeled prion protein. Bunches of multi-charged peaks were then analyzed and deconvoluted manually. For deconvolution, non linear regression analysis with equation (1) was used:
(1)
where m/z stands for mass to charge ratio, which is detected directly by ESI MS; z stands for charge of the ion; and M is a parameter of equation, which represents molecular weight of the prion protein.
ESI spectra and deconvolution fits can be seen further.
After deconvolution of the highest peaks of the unlabeled prion protein, we obtained: M = 25095(correlation coefficient R2 = 1.000000, = 0.00135). Theoretical average molecular mass is M = 25095.35.
ESI of the full-length unlabeled prion protein 2g with the values of the highest peaks
Deconvolution fit of the full-length unlabeled prion protein 2g
After deconvolution of the highest peaks of the labeled prion protein, we obtained: M = 25579 (correlation coefficient R2 = 1.000000, = 0.00509). Theoretical average molecular mass is M = 25578.70.
ESI of the full-length labeled prion protein 4g with the values of the highest peaks
Deconvolution fit of full-length labeled prion protein 4g