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Comparative analysis of peptides and proteins in two congeneric (Leiurus abdullahbayrami; Buthidae ) scorpion venoms by MALDI-TOFMS

Year 2022, Volume 15, Issue 2, 186 - 194, 15.08.2022
https://doi.org/10.46309/biodicon.2022.1095531

Abstract

In this study, two Leiurus abdullahbayrami scorpion venoms were analyzed wif MALDI-TOFMS for teh first time and their peptide and protein distributions were compared. Teh total protein amount of teh venoms was measured wif teh Nano Ready Touch device at a wavelength of 280 and was prepared to be equal to 2mg/mL concentration. Afterwards, teh supernatants were separated by centrifugation at +4 ºC for 15 minutes at 15,000 rpm, and 250 µL of matrix solution (18 mg/mL α-CHCA, 1:1; v/v) was added and scorpion venom matrix (AVMx) samples were added. has been prepared. For analysis wif MALDI-TOFMS (Microflex-LT), parallel spots (1µL) from AVMx samples of both scorpion venoms were placed on teh steel plate, and loaded into teh device after drying at room temperature. Teh system was operated in linear positive ion mode in teh mass range of 1-50 kDa, and a 60 Hz nitrogen laser at 337 nm was used as teh ion source. Mass spectra were generated using teh flexAnalysis software (version 3.4). All data were generated virtual gel images containing teh projection of teh peaks in teh venom spectra using teh filo-proteomic TEMPprincipal component analysis (PCA) method supported by teh built-in MATLAB software integrated into teh MALDI Biotyper software (version 3.1). As a result, it has been experimentally determined dat teh relative ratios of peptide and protein molecules and venoms may vary in scorpions belonging to teh same species. On teh other hand, it has been observed dat some basic peptide structures (3555; 3772, 3996, 6780, 6820 Da; m/z) of these creatures, which preserve their species characteristics against evolution, remain teh same.

References

  • [1] Rincón-Cortés, C.A., Olamendi-Portugal T., Carcamo-Noriega E.N., Santillán E.G., Zuñiga F.Z.,& Reyes-Montaño, E.A. (2019). Structural and Functional Characterization of Toxic Peptides Purified from the Venom of the Colombian Scorpion Tityus Macrochirus. Toxicon, 169, 5-11. https://doi.org/10.1016/j.toxicon.2019.07.013.
  • [2] Salama, W.M. & Sharshar, K.M. (2013). Surveillance study on scorpion species in Egypt and comparison of their crude venom protein profiles. J. Basic Appl. Zool., 66(2), 76-86. doi: 10.1016/j.jobaz.2013.10.003.
  • [3] Schaffrath, S. & Predel, R. (2014). A simple protocol for venom peptide barcoding in scorpions. EuPA Open Proteomics, 3, 239-245. doi: 10.1016/j.euprot.2014.02.017.
  • [4] Oukkache, N., Rossoa, J.P., Alamia, M., Ghalimb, N., Sailec, R., Mohammed, H.,et.al. (2008). New analysis of the toxic compounds from the Androctonus mauretanicus mauretanicus scorpion venom. Toxicon, 51(5), 835-852. doi: 10.1016/j.toxicon.2007.12.012.
  • [5] Yağmur, E.A., Koç, H., & Kunt, K.B. (2009). Description of a new species of Leiurus Ehrenberg, 1828 (Scorpiones: Buthidae) from Southеastеrn Turkey. Euscorpius, 85, 1-20. doi: 10.18590/euscorpius.2009.vol2009.iss85.1.
  • [6] Ulutaş, V. (2017). Leiurus abdullahbayrami Türü Akrep Venomunun Proteomik Analizi (Y.lisans tezi). Biyoteknoloji ve Biyogüvenlik Anabilim Dalı, Eskişehir Osmangazi Üniversitesi Fen Bilimleri Enstitüsü.
  • [7] Çalişkan, F., Sivas, H. & Şahin, Y. (2009). A preliminary study for the detection of gelatinolytic proteases from the scorpion Androctonus crassicauda (Turkish black scorpion) venom. Turkish J. Biochem., 34(3), 148-153.
  • [8] Newton, K.A., Clench, M R., Deshmukh, R., Jeyaseelan, K., & Strong, P. N. (2007). Mass fingerprinting of toxic fractions from the venom of the Indian red scorpion, Mesobuthus tamulus: biotope-specific variation in the expression of venom peptides. Rapid Commun. Mass Spectrom., 21(21), 3467-3476. doi: 10.1002/rcm.3240.
  • [9] Borges, A. & Rojas-Runjaic, F.J.M. (2007). Tityus perijanensis González-Sponga (Scorpiones, Buthidae): Molecular assessment of its geographical distribution and venom lethality of Venezuelan populations. Toxicon, 50(7), 1005-1010. doi: 10.1016/j.toxicon.2007.07.019.
  • [10] Estrada-Gómez, S., Vargas-Muñoz, L.J.M, Saldarriaga-Córdoba, M. & van der Meijden, A. (2021). MS/MS analysis of four scorpion venoms from Colombia: A descriptive approach. J. Venom. Anim. Toxins Incl. Trop. Dis., 27, 1-13. doi: 10.1590/1678-9199-JVATITD-2020-0173.
  • [11] Bringans, S., Eriksen, S., Kendrick, T., Gopalakrishnakone, P., Livk1, A., Lock, R. (2008). Proteomic analysis of the venom of Heterometrus longimanus (Asian black scorpion). Proteomics, 8(5), 1081-1096. doi: 10.1002/pmic.200700948.
  • [12] Nascimento, D.G., Rates, B., Santos, D.M., - Braga, T.V., Barbosa-Silva, A., & Dutra A.A.A. et al., (2006). Moving pieces in a taxonomic puzzle: Venom 2D-LC/MS and data clustering analyses to infer phylogenetic relationships in some scorpions from the Buthidae family (Scorpiones). Toxicon, 47(6), 628-639. doi: 10.1016/j.toxicon.2006.01.015.
  • [13] Favreau, P., Menin, L., Michalet, S., Perret, F., Cheneval, O.,& Sto¨cklin, M., et al. (2006). Mass spectrometry strategies for venom mapping and peptide sequencing from crude venoms: Case applications with single arthropod specimen. Toxicon, 47(6), 676-687. doi: 10.1016/j.toxicon.2006.01.020.
  • [14] Martin-Eauclaire, M.F., Granjeaud, S., Belghazi, M., & Bougis, P.E. (2013). Achieving automated scorpion venom mass fingerprinting (VMF) in the nanogram range. Toxicon, 69, 211-218. doi: 10.1016/j.toxicon.2013.03.001.
  • [15] Schaffrath, S., & Predel, R. (2014). A simple protocol for venom peptide barcoding in scorpions. EuPA Open Proteomics, 3, 239-245. doi: 10.1016/j.euprot.2014.02.017.
  • [16] Abbas, N.M., Belghazi, Y., Abdel-Mottaleb, J., Tytgat, P., Bougis, E., & Martin-Eauclaire, M.F.( 2008). A new Kaliotoxin selective towards Kv1.3 and Kv1.2 but not Kv1.1 channels expressed in oocytes. Biochem. Biophys. Res. Commun., 376(3), 525-530. doi: 10.1016/j.bbrc.2008.09.033.
  • [17] Sauget, M., Valot, B., Bertrand, X. & Hocquet, D. (2017). Can MALDI-TOF Mass Spectrometry Reasonably Type Bacteria?. Trends Microbiol., 25(6), 447-455. doi: 10.1016/j.tim.2016.12.006.
  • [18] Smith, J.J., Jones, A. & Alewood, P.F. (2012). Mass landscapes of seven scorpion species: The first analyses of Australian species with 1,5-DAN matrix. J. Venom Res., 3, 7-14.
  • [19] Ozkan, O., Yagmur, E.A. & Ark, M. (2011). A newly described scorpion species, leiurus abdullahbayrami (Scorpion: Buthidae), and the lethal potency and in vivo effects of its venom. J. Venom. Anim. Toxins Incl. Trop. Dis., 17(4), 414-421. doi: 10.1590/S1678-91992011000400008.
  • [20] Erdeş, E., Doğan, T.S., Coşar, İ., Danışman, T., Kunt, K.B,& Şeker, T., et al., (2014). Characterization of Leiurus abdullahbayrami (Scorpiones: Buthidae) venom: peptide profile, cytotoxicity and antimicrobial activity. J. Venom. Anim. Toxins Incl. Trop. Dis., 20(1), 48. doi: 10.1186/1678-9199-20-48.
  • [21] Samad, R.A. & Al Disi, Z. (2020). The use of principle component analysis and MALDI-TOF MS for the di ff erentiation of mineral forming Virgibacillus and Bacillus species isolated from sabkhas. R. Soc. Chem., 10, 14606-14616. doi: 10.1039/d0ra01229g.
  • [22] Jeong, Y., Lee, J. & Kim, S. (2013). Discrimination of Bacillus anthracis Spores by Direct in-situ Analysis of Matrix-Assisted Laser Desorption / Ionization Time-Of-Flight Mass Spectrometry. 34(9), 2635-2639.
  • [23] Ducancel, F. (2016). Les venins au service de la recherche médicale. Biol. Aujourdhui., 210(2), 89-99. doi: 10.1051/jbio/2016016.
  • [24] Pucca, M.B., Ahmadi, S., Cerni, F.A., Ledsgaard, L., Sørensen, C.V.,& McGeoghan, F.T.S. et al. (2020). Unity Makes Strength: Exploring Intraspecies and Interspecies Toxin Synergism between Phospholipases A2 and Cytotoxins. Front. Pharmacol., 11. doi: 10.3389/fphar.2020.00611. [25] Morgenstern, D., & King, G.F. (2013). The venom optimization hypothesis revisited. Toxicon, 63(1), 120-128. doi: 10.1016/j.toxicon.2012.11.022.

MALDI-TOFMS ile iki türdeş (Leiurus abdullahbayrami; Buthidae) akrep zehirindeki peptit ve proteinlerin karşılaştırmalı analizi

Year 2022, Volume 15, Issue 2, 186 - 194, 15.08.2022
https://doi.org/10.46309/biodicon.2022.1095531

Abstract

Bu çalışmada, iki Leiurus abdullahbayrami akrep venomunun ilk defa MALDI-TOFMS ile analizleri yapılarak, peptit ve protein dağılımları karşılaştırılmıştır. Venomların toplam protein miktarı Nano Ready Touch cihazı ile 280 dalga boyunda ölçülmüş ve 2mg/mL konsantrasyona eşit olacak şekilde hazırlanmıştır. Daha sonra +4 ºC'de 15.000 rpm'de 15 dakika santrifüj edilerek süpernatantlar ayrıldıktan sonra üzerine 250 µL matriks çözeltisi (18 mg/mL α-CHCA, 1:1; v/v) ilave edilmiş ve akrep venom matriks örnekleri hazırlanmıştır. MALDI-TOFMS (Microflex-LT) ile analizleri meçin çelik plaka üzerine her iki akrep venomuna ait AVMx numunesinden paralel olarak spotlanmış (1µL), oda sıcaklığında kurutulduktan sonra cihaza yüklenmiştir. Sistem 1-50 kDa kütle aralığında lineer pozitif iyon modunda çalıştırılarak iyon kaynağı olarak 337 nm'de 60 Hz nitrojen lazer kullanılmıştır. Kütle spektrumları flexAnalysis yazılımı (versiyon 3.4) kullanılarak oluşturulmuştur. Tüm veriler, MALDI Biotyper yazılımına (3.1 sürüm) entegre edilmiş yerleşik MATLAB yazılımı tarafından desteklenen filo-proteomik temel bileşen analizi (TBA) yöntemi ile venom spektrumlarındaki piklerin izdüşümünü bulunduran sanal jel görüntüleri oluşturulmuştur. Sonuç olarak, peptit ve protein moleküllerinin ve venomlardaki bağıl oranlarının aynı türe ait akreplerde de değmeşkenlik gösterebileceğme deneysel olarak tespit edilmiştir. Öte yandan evrimleşmeye karşı tür karakteristiğini koruyan bu canlıların bazı temel peptit yapılarının (3555; 3772, 3996, 6780, 6820 Da; m/z) aynı kaldığı da gözlenmiştir.

References

  • [1] Rincón-Cortés, C.A., Olamendi-Portugal T., Carcamo-Noriega E.N., Santillán E.G., Zuñiga F.Z.,& Reyes-Montaño, E.A. (2019). Structural and Functional Characterization of Toxic Peptides Purified from the Venom of the Colombian Scorpion Tityus Macrochirus. Toxicon, 169, 5-11. https://doi.org/10.1016/j.toxicon.2019.07.013.
  • [2] Salama, W.M. & Sharshar, K.M. (2013). Surveillance study on scorpion species in Egypt and comparison of their crude venom protein profiles. J. Basic Appl. Zool., 66(2), 76-86. doi: 10.1016/j.jobaz.2013.10.003.
  • [3] Schaffrath, S. & Predel, R. (2014). A simple protocol for venom peptide barcoding in scorpions. EuPA Open Proteomics, 3, 239-245. doi: 10.1016/j.euprot.2014.02.017.
  • [4] Oukkache, N., Rossoa, J.P., Alamia, M., Ghalimb, N., Sailec, R., Mohammed, H.,et.al. (2008). New analysis of the toxic compounds from the Androctonus mauretanicus mauretanicus scorpion venom. Toxicon, 51(5), 835-852. doi: 10.1016/j.toxicon.2007.12.012.
  • [5] Yağmur, E.A., Koç, H., & Kunt, K.B. (2009). Description of a new species of Leiurus Ehrenberg, 1828 (Scorpiones: Buthidae) from Southеastеrn Turkey. Euscorpius, 85, 1-20. doi: 10.18590/euscorpius.2009.vol2009.iss85.1.
  • [6] Ulutaş, V. (2017). Leiurus abdullahbayrami Türü Akrep Venomunun Proteomik Analizi (Y.lisans tezi). Biyoteknoloji ve Biyogüvenlik Anabilim Dalı, Eskişehir Osmangazi Üniversitesi Fen Bilimleri Enstitüsü.
  • [7] Çalişkan, F., Sivas, H. & Şahin, Y. (2009). A preliminary study for the detection of gelatinolytic proteases from the scorpion Androctonus crassicauda (Turkish black scorpion) venom. Turkish J. Biochem., 34(3), 148-153.
  • [8] Newton, K.A., Clench, M R., Deshmukh, R., Jeyaseelan, K., & Strong, P. N. (2007). Mass fingerprinting of toxic fractions from the venom of the Indian red scorpion, Mesobuthus tamulus: biotope-specific variation in the expression of venom peptides. Rapid Commun. Mass Spectrom., 21(21), 3467-3476. doi: 10.1002/rcm.3240.
  • [9] Borges, A. & Rojas-Runjaic, F.J.M. (2007). Tityus perijanensis González-Sponga (Scorpiones, Buthidae): Molecular assessment of its geographical distribution and venom lethality of Venezuelan populations. Toxicon, 50(7), 1005-1010. doi: 10.1016/j.toxicon.2007.07.019.
  • [10] Estrada-Gómez, S., Vargas-Muñoz, L.J.M, Saldarriaga-Córdoba, M. & van der Meijden, A. (2021). MS/MS analysis of four scorpion venoms from Colombia: A descriptive approach. J. Venom. Anim. Toxins Incl. Trop. Dis., 27, 1-13. doi: 10.1590/1678-9199-JVATITD-2020-0173.
  • [11] Bringans, S., Eriksen, S., Kendrick, T., Gopalakrishnakone, P., Livk1, A., Lock, R. (2008). Proteomic analysis of the venom of Heterometrus longimanus (Asian black scorpion). Proteomics, 8(5), 1081-1096. doi: 10.1002/pmic.200700948.
  • [12] Nascimento, D.G., Rates, B., Santos, D.M., - Braga, T.V., Barbosa-Silva, A., & Dutra A.A.A. et al., (2006). Moving pieces in a taxonomic puzzle: Venom 2D-LC/MS and data clustering analyses to infer phylogenetic relationships in some scorpions from the Buthidae family (Scorpiones). Toxicon, 47(6), 628-639. doi: 10.1016/j.toxicon.2006.01.015.
  • [13] Favreau, P., Menin, L., Michalet, S., Perret, F., Cheneval, O.,& Sto¨cklin, M., et al. (2006). Mass spectrometry strategies for venom mapping and peptide sequencing from crude venoms: Case applications with single arthropod specimen. Toxicon, 47(6), 676-687. doi: 10.1016/j.toxicon.2006.01.020.
  • [14] Martin-Eauclaire, M.F., Granjeaud, S., Belghazi, M., & Bougis, P.E. (2013). Achieving automated scorpion venom mass fingerprinting (VMF) in the nanogram range. Toxicon, 69, 211-218. doi: 10.1016/j.toxicon.2013.03.001.
  • [15] Schaffrath, S., & Predel, R. (2014). A simple protocol for venom peptide barcoding in scorpions. EuPA Open Proteomics, 3, 239-245. doi: 10.1016/j.euprot.2014.02.017.
  • [16] Abbas, N.M., Belghazi, Y., Abdel-Mottaleb, J., Tytgat, P., Bougis, E., & Martin-Eauclaire, M.F.( 2008). A new Kaliotoxin selective towards Kv1.3 and Kv1.2 but not Kv1.1 channels expressed in oocytes. Biochem. Biophys. Res. Commun., 376(3), 525-530. doi: 10.1016/j.bbrc.2008.09.033.
  • [17] Sauget, M., Valot, B., Bertrand, X. & Hocquet, D. (2017). Can MALDI-TOF Mass Spectrometry Reasonably Type Bacteria?. Trends Microbiol., 25(6), 447-455. doi: 10.1016/j.tim.2016.12.006.
  • [18] Smith, J.J., Jones, A. & Alewood, P.F. (2012). Mass landscapes of seven scorpion species: The first analyses of Australian species with 1,5-DAN matrix. J. Venom Res., 3, 7-14.
  • [19] Ozkan, O., Yagmur, E.A. & Ark, M. (2011). A newly described scorpion species, leiurus abdullahbayrami (Scorpion: Buthidae), and the lethal potency and in vivo effects of its venom. J. Venom. Anim. Toxins Incl. Trop. Dis., 17(4), 414-421. doi: 10.1590/S1678-91992011000400008.
  • [20] Erdeş, E., Doğan, T.S., Coşar, İ., Danışman, T., Kunt, K.B,& Şeker, T., et al., (2014). Characterization of Leiurus abdullahbayrami (Scorpiones: Buthidae) venom: peptide profile, cytotoxicity and antimicrobial activity. J. Venom. Anim. Toxins Incl. Trop. Dis., 20(1), 48. doi: 10.1186/1678-9199-20-48.
  • [21] Samad, R.A. & Al Disi, Z. (2020). The use of principle component analysis and MALDI-TOF MS for the di ff erentiation of mineral forming Virgibacillus and Bacillus species isolated from sabkhas. R. Soc. Chem., 10, 14606-14616. doi: 10.1039/d0ra01229g.
  • [22] Jeong, Y., Lee, J. & Kim, S. (2013). Discrimination of Bacillus anthracis Spores by Direct in-situ Analysis of Matrix-Assisted Laser Desorption / Ionization Time-Of-Flight Mass Spectrometry. 34(9), 2635-2639.
  • [23] Ducancel, F. (2016). Les venins au service de la recherche médicale. Biol. Aujourdhui., 210(2), 89-99. doi: 10.1051/jbio/2016016.
  • [24] Pucca, M.B., Ahmadi, S., Cerni, F.A., Ledsgaard, L., Sørensen, C.V.,& McGeoghan, F.T.S. et al. (2020). Unity Makes Strength: Exploring Intraspecies and Interspecies Toxin Synergism between Phospholipases A2 and Cytotoxins. Front. Pharmacol., 11. doi: 10.3389/fphar.2020.00611. [25] Morgenstern, D., & King, G.F. (2013). The venom optimization hypothesis revisited. Toxicon, 63(1), 120-128. doi: 10.1016/j.toxicon.2012.11.022.

Details

Primary Language Turkish
Subjects Biochemistry and Molecular Biology
Journal Section Research Articles
Authors

Yasemin NUMANOĞLU ÇEVİK> (Primary Author)
T.C.S.B. TÜRKİYE HALK SAĞLIĞI GENEL MÜDÜRLÜĞÜ
0000-0001-5818-7881
Türkiye


Mehmet Ali KANAT>
T.C. S.B. Halk Sağlığı Genel Müdürlüğü,
0000-0003-3494-4563
Türkiye

Thanks THSGM-ASÜL’e sağılmış akrep venom numunelerini laboratuvarımıza hibe ettiği için çok müteşekkiriz.
Early Pub Date August 3, 2022
Publication Date August 15, 2022
Application Date March 31, 2022
Acceptance Date July 12, 2022
Published in Issue Year 2022, Volume 15, Issue 2

Cite

APA Numanoğlu Çevik, Y. & Kanat, M. A. (2022). MALDI-TOFMS ile iki türdeş (Leiurus abdullahbayrami; Buthidae) akrep zehirindeki peptit ve proteinlerin karşılaştırmalı analizi . Biyolojik Çeşitlilik ve Koruma , 15 (2) , 186-194 . DOI: 10.46309/biodicon.2022.1095531

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