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

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.

Keywords

• Scorpion
• venom
• Leiurus Abdullahbayrami
• MALDI-TOF MS
• Mass spectrometry

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

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.

Keywords

• Akrep
• venom
• Leiurus Abdullahbayrami
• MALDI-TOFMS
• Kütle spetrometresi

Thanks

THSGM-ASÜL’e sağılmış akrep venom numunelerini laboratuvarımıza hibe ettiği için çok müteşekkiriz.

References

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