Venomics and Peptidomics of Palearctic Vipers: A Clade-Wide Analysis of Seven Taxa of the Genera Vipera, Montivipera, Macrovipera, and Daboia across Türkiye

Snake venom variations are a crucial factor to understand the consequences of snakebite envenoming worldwide, and therefore it is important to know about toxin composition alterations between taxa. Palearctic vipers of the genera Vipera, Montivipera, Macrovipera, and Daboia have high medical impacts across the Old World. One hotspot for their occurrence and diversity is Türkiye, located on the border between continents, but many of their venoms remain still understudied. Here, we present the venom compositions of seven Turkish viper taxa. By complementary mass spectrometry-based bottom-up and top-down workflows, the venom profiles were investigated on proteomics and peptidomics level. This study includes the first venom descriptions of Vipera berus barani, Vipera darevskii, Montivipera bulgardaghica albizona, and Montivipera xanthina, as well as the first snake venomics profiles of Turkish Macrovipera lebetinus obtusa, and Daboia palaestinae, including an in-depth reanalysis of M. bulgardaghica bulgardaghica venom. Additionally, we identified the modular consensus sequence pEXW(PZ)1–2P(EI)/(KV)PPLE for bradykinin-potentiating peptides in viper venoms. For better insights into variations and potential impacts of medical significance, the venoms were compared against other Palearctic viper proteomes, including the first genus-wide Montivipera venom comparison. This will help the risk assessment of snakebite envenoming by these vipers and aid in predicting the venoms’ pathophysiology and clinical treatments.


Addi onal Materials and Methods
Detailed Bo om-up proteomics -Snake Venomics:
Peaks later than 25 min were further processed by the snake venomics steps of gel separa on and tryp c digest, peaks with earlier reten on mes (Rt) are known for their low molecular mass pep de content and were directly sent to the LC-MS.The viperine abundant tripep de pEKW (with pE for pyroglutamate) signal at around 25 min was set as benchmark.

SDS-PAGE profiling and tryp c diges on
The dried venom frac ons were redissolved in 10 µL reducing 2× sodium dodecyl sulfate (SDS) sample buffer (125 mM Tris HCl pH 6.8, 4% (w/v) SDS, 17.5% (w/v) glycerol, 0.02% (w/v) Bromphenol blue and 200 mM freshly prepared dithiothreitol DTT in ultra-pure (MQ) water), heated for 10 min at 95 °C, fully loaded and separated using a 12% SDS-PAGE (SurePage Bis-Tris, Genscript, Piscataway, NJ, USA) run with MES buffer (50 mM 2-(N-morpholino)ethane sulfonic acid (MES), 50 mM Tris base, 1 mM EDTA, 0.1% (w/v) SDS, stored in brown glass flasks at 4°C) at 200 V for 21 min.A PageRuler Unstained Protein Ladder (Thermo Scien fic, Waltham, MA, USA) was used as protein mass standard.Gels were three mes short-washed with water.Proteins were fixed with preheated (50-60 °C) fixa on buffer three mes for 10 min each (aqueous, 40% (v/v) methanol, 10% (v/v) ace c acid), stained for 45 min in preheated (50-60 °C) fast staining buffer (aqueous, 0.3% (v/v) HCl 37%, 100 mg/L Coomassie 250G) under constant mild shaking, and kept overnight at 4 °C in storage buffer (aqueous, 20% (v/v) methanol, 10% (v/v) ace c acid) for destaining.The cleaned gels were then scanned for documenta on and quan fica on.Gel pieces with single protein bands were cut, dried with 500 µL ACN, and stored at −20 °C without ACN un l tryp c diges on.The disulfide bridges were reduced with 30 µL freshly prepared DTT (100 mM in 100 mM ammonium hydrogen carbonate (ABC) per gel band) for 30 min at 56 °C and dried with 500 µL ACN for 10 min before removing the supernatant.Cysteines were alkylated with freshly prepared iodoacetamide (55 mM in 100 mM ABC) for 20 min at room temperature in the dark to protect the reduced thiols from oxida on and washed with 500 µL ACN for 2 min.before removing the supernatant.Gel samples were dried again with 500 µL ACN for 15 min, ACN removed, followed by 30 min incuba on on ice with 30 µL freshly ac vated trypsin (13.3 ng/µL, 10% (v/v) ACN in 10 mM ABC; Pierce trypsin, Thermo, Rockfeld, IL, USA).When necessary, addi onal volumes of trypsin were added, so that the gel piece was s ll covered in buffer.All samples were incubated for 90 min on ice, 20 µL ABC buffer (10 mM) was added, and were incubated overnight at 37 °C.Pep des were extracted with 100 µL pre-warmed elu on buffer (aqueous, 30% (v/v) ACN MS grade, 5% (v/v) HFo) at 37 °C for 30 min.The supernatant was transferred into a separate microtube, vacuum-dried and if possible directly prepared for the LC-MS/MS measurement, else samples were stored at −20 °C.

Detailed Bo om-up proteomics -Mass Spectrometry:
The following gradient with ultrapure water with 0.1% (v/v) HFo (solvent A) and ACN with 0.1% (v/v) HFo (solvent B) was used at 0.3 mL/min, with a linear gradient between the me points, given at min (B%): 0-1 (5% const.),1-11 (5 to 40%), 11-12 (40 to 99%), 12-13 (99% const.), and 2 min reequilibra on at 5% B. The parameters in the ESI posi ve modus were as follows: 270 °C capillary temperature, 45 L/min sheath gas, 10 L/min auxiliary gas, 4.0 kV source voltage, 100.0 µA source current, 20 V capillary voltage, 130 V tube lens.FTMS measurements were performed with 1 μ scans and 1000 ms maximal fill me.AGC targets were set to 10 6 for full scans and to 3 × 10 5 for MS2 scans.MS2 scans were performed with a mass resolu on (R) of 60,000 (at m/z 400) for m/z 250-2000.MS2 spectra were obtained in data-dependent acquisi on (DDA) mode as top2 with 35 V normalized CID energy, and 500 as the minimal signal required with an isola on width of 3.0.The default charge state was set to z = 2, and the ac va on me to 30 ms.Unassigned charge states and charge state 1 were rejected for tryp c digest pep des, for direct submi ed frac ons from the ini al HPLC run all charge states were measured.

Detailed Top-down proteomics -Mass Spectrometry:
For the denaturing TD analysis, 100 µg of lyophilized venom was dissolved to a final concentra on of 10 mg/mL in aqueous 1% (v/v) HFo and centrifuged for 5 min at 20,000 × g.The supernatant was mixed with 30 μL of citrate buffer (0.1 M, pH 3.0) and split into two aliquots.The first aliquot was mixed 10 μL of 0.5 M tris(2-carboxyethyl)phosphine (TCEP), for reduc on of disulfide bonds, and incubated for 30 min at 65 °C.The second was supplemented with 10 μL of ultrapure water and will be referred as non-reduced sample.Both samples were centrifuged for 5 min at 20,000 × g and 10 µL of each was injected into an Q Exac ve HF mass spectrometer (Thermo, Bremen, Germany) via a Vanquish ultrahigh performance liquid chromatography (UHPLC) system (Agilent Technologies, Waldbronn, Germany) using a reversed-phase Supelco Discovery BIO wide C18 (2.0 × 150 mm; 3 μm par cle size; 300 Å pore size) column thermostated at 30 °C.The following gradient with ultrapure water with 0.1% (v/v) HFo (solvent A) and ACN with 0.1% (v/v) HFo (solvent B) was used at 0.4 mL/min, with a linear gradient between the me points, given at min (B%): 0-6 (5% const.),6-25 (5 to 40%), 25-30 (40 to 70%), 30-35 (70% const.), and 5 min re-equilibra on at 5% B. The parameters in the ESI posi ve modus were as follows: 265.50 °C capillary temperature, 50.00 AU sheath gas, 12.50 L/min auxiliary gas, 3.50 kV source voltage, 100.00 µA source current.FTMS measurements were performed with 1 μ scans and 1000 ms maximal fill me.MS2 scans were performed with a mass resolu on (R) of 140,000 (at m/z 200).MS2 spectra were obtained in DDA mode as top3 with 30% normalized high energy C-trap dissocia on (HCD) and an isola on window of m/z 3.0.The default charge state was set to z = 6, and the ac va on me to 30 ms.Unassigned charge states and isotope states were rejected for MS2 measurements.

Detailed proteome quan fica on:
The comparable approach determine a toxin family abundance in the venom as the sum of all its normalized toxin abundances T: The normalized toxin abundance within a single protein band Tband is calculated with the normalized values of the RP-HPLC peak integral P measured at 214 nm, the densitometric gel band intensity D and if necessary the rela ve MS ion intensity M of the most abundant and iden fied pep des: For the peak quan fica on a er blank run subtrac on, the HPLC separa on chromatogram frac ons were integrated as area under the curve Ppeak in ra o to the total sum of all peaks: = ∑ For the densitometric quan fica on of a single SDS band, the non-highly compressed gel scan (here in PNG format) was processed by Fiji 82 .The colour depth was set to 8bit grayscale and inverted to integrate former darker bands with higher values.The band area Aband and the corresponding integrated band densi es Dband were measured for each band, as well as a corresponding background areas Abg and integrated band densi es Dbg.By removing the propor on of background, we calculated the normalized gel band intensity D for each toxin band in the gel: In case of mul ple toxin iden fica on within a single band, single normalized toxin abundances M were es mated based on the ion intensity sum of the three most intensive pep de ions of one toxin from M3 in rela on to the sum of all top3 toxin ions from the other co-migrated toxins families within this MS sample, as summarised in Calvete et al. 2023: In total, band iden fica on based on the BU, TD and pep domics results, in comparison to the IMP and the apparent masses of the SDS bands.

Table S8 : Quantification of the M. l. obtusa venom pool proteome.
*