- Proteomic characterization of two snake venoms: Naja naja atra and Agkistrodon halys

Proteomic strategy to analyse snake venom proteomes
In spite of extensive developments in proteomic techniques, there is not a panacea in the field. One method may have strong advantages for the analysis of some proteomes, but may have disadvantages for others. A major concern for the proteomic analysis of snake venom is the variable range of molecular sizes of the proteins and peptides. To alleviate this problem, we have adopted two strategies: (i) utilizing techniques such as shotgun-LC-MS/MS and 1DE-LC-MS/MS, whereby direct tryptic digestion or electrophoresis could minimize loss of proteins and peptides, and (ii) using GF to separate small venom peptides from the larger proteins; the separated proteins and peptides treated further by 2DE or HPLC lead to better resolution for venom components. The experimental procedure is depicted in Scheme 1. Two techniques of MS, MALDI–TOF MS and LC-MS/MS, were used for protein identification in these experiments. In contrast with MALDI–TOF MS, LC-MS/MS offers the advantages of reverse-phase HPLC for more extensive separation of peptide mixtures, and ion-trap for better resolution of peptide sequences. Thus we conducted protein identification of 2DE gel spots using MALDI–TOF MS, but, in most cases, LC-MS/MS was the primary means for protein identification.


Comparison of snake venom proteomes using the shotgun digestion approach
A typical ion chromatogram for the tryptic digestion of Agki venom proteins is shown in Figure 1. The peptide chromatogram monitored at 214 nm in Figure 1(A) indicates that the loaded peptides are completely eluted at an acetonitrile concentration of 80%. The reproducibility of the identified proteins was approx. 96% among parallel experiments (three injections/sample). A full-scan mass spectrum from LC-MS/MS analysis of Agki venom is shown in Figure 1(B), a single MS survey scan at elution time of 82.13 min is represented in Figure 1(C) and a MS/MS scan of the precursor ion with a m/z value of 567.11 triggered by the survey scan is depicted in Figure 1(D). Depending on the population of the constituent peptides, approx. 4000 MS/MS spectra were automatically collected for each injection of Naja or Agki venom. After acquisition of MS/MS spectra, the proteins or peptides were identified by the software algorithm Mascot. Of the spectra, approx. 10% gave reliable candidate peptides by searching NCBI and EBI protein databases of snake venom. A total of 78 and 28 unique proteins or peptides were identified from Naja and Agki venom respectively. All of the unique proteins were determined by more than two peptides matched. The information regarding the identified proteins, such as the matched peptide sequences, accession number, theoretical pI and MM, their charge states, and m/z values, are given in Supplementary Table I (available at
To compare the biological functions of the identified proteins in this study, venomous proteins or peptides are broadly classified into three toxin groups classified by their toxicity to animals: (i) neurotoxin, (ii) cardiotoxin and (iii) haemotoxin. Two major protein families, phospholipase A2 or metalloproteinase, generally exist in snake venoms; they are hence listed as specific categories in this study. It is worth noting that the delineation of protein categories is not, in every case, clear cut due to the multiple toxic attributes of proteins.

According to the classification described above, the distributions of the identified proteins in five categories are listed in Table 1. As expected, Naja venom contains high amounts of cardio- and neuro-toxins, both occupying approx. 60% of the composition, and Agki has a high abundance of proteins involved in disruption of the haemostatic system, with 28% of haemotoxins and 40% of metalloproteinases. The content of phospholipase A2 in both venoms is close, at 18% and 14% respectively. Although some identified proteins have diverse amino acid sequences, these proteins may belong to the same protein family, at least performing similar biological functions. For instance, five unique proteins identified in Naja by this approach are assigned as GI numbers 1000502, 1326087, 1661022, 1134871 and 299268 that are the members of cardiotoxin 1 family with over 80% identity of amino acid sequence (see Supplementary Tables at

Comparison of snake venom proteomes using a 1DE-LC-MS/MS approach
Figure 2 shows 15% polyacrylamide gels that were loaded and electrophoresed with 300 μg of snake venom proteins. As shown, the major components of Naja venom are distributed at a MM lower than 10 kDa, whereas Agki venom proteins are spread evenly through a wide range of MMs. To separate the proteins effectively, each SDS/PAGE gel was evenly divided into ten slices, approx. 5 mm/slice, and subsequently digested by trypsin and separated by reverse-phase HPLC followed by analysis of tandem mass spectrometry. The collected MS/MS spectra varied from 500 to 2000/slice. In total, 5500 and 9000 MS/MS spectra were collected for Naja and Agki venom respectively. The spectra matched 65 and 43 unique proteins or peptides for Naja and Agki venom respectively (see details in Supplementary Table I at

These identified venomous proteins or peptides are categorized in Table 1. In Naja venom, 74% of the identified proteins and peptides are cardiotoxins and neurotoxins, but only one haemotoxin was found in this venom. In contrast with Naja, Agki venom has 23% haemotoxin and 30% metalloproteinases, but only three cardiotoxins were detected, in which cardiotoxin-2c (GI number 3342766) is commonly found in both venoms. Comparing the differences between theoretical and apparent values of MM, 94% of the proteins identified from Naja venom display good correlations, whereas 46% of Agki proteins closely matched their theoretical predictions. Over 50% of Agki proteins migrated on SDS/PAGE with significantly low apparent MMs, suggesting that the degradation caused by proteases is common in Agki venom. Interestingly, none of the phospholipases A2 in Agki were found degraded; on the contrary, more than 80% of metalloproteinases appeared as the reduced molecular sizes on SDS/PAGE.

Comparison of snake venom proteomes using GF-LC-MS/MS and GF-2DE-MALDI–TOF-MS approaches
The approach of 2DE is a powerful technique to separate and identify soluble proteins; however, it is limited to the analysis of small proteins and peptides. Therefore size-exclusion chromatography was employed to separate venom proteins into two crude fractions, small proteins or peptides (MM10 kDa). The small proteins were subjected directly to tryptic digestion, and the large proteins were separated further by 2DE. As shown in Figure 3, the eluted fractions from Sephadex G-50 GF were examined by SDS/PAGE, in which the venomous proteins are roughly separated according to their molecular sizes. Obviously, the protein profile of Naja venom is different from that of Agki, owing to the abundance of small proteins and peptides eluted in later fractions. The initial fractions containing the large proteins (>10 kDa) were pooled and subjected to 2DE (1 mg of protein/gel). As 2DE separation of venomous proteins shows in Figure 4, the striking difference between the two venom samples is the distribution pattern of proteins along the horizontal pH range. Notably, most proteins from the Naja sample migrated to basic regions (pH>7), whereas most of the Agki venom proteins were located in the acidic pH region (pH

The fractions containing proteins with MMNaja and Agki venom respectively. Of these spectra, approx. 10% gave reliable candidate peptides by searching the snake venom protein database. Totals of 77 and 48 unique proteins or peptides were confirmed for Naja and Agki venom respectively (Table 1; details in Supplementary Table I at The proteins with apparent sizes more than 10 kDa were analysed by 2DE and subjected to MALDI–TOF-MS after staining by Coomassie Brilliant Blue and in-gel digestion as described above. A mass fingerprint of the unfractionated peptide mixture was obtained by MALDI–TOF-MS followed by Mascot protein identification mapping. To ensure the accuracy of protein identification, the following criteria were followed for each sample: (i) ranked in top two hits; (ii) at least four matched sequences; and (iii) over 10% protein sequence coverage. Totals of 190 and 169 spots from Naja and Agki respectively were picked from the corresponding gel. For Naja venom, 152 high-quality MALDI–TOF-MS spectra were collected and 100 spectra were matched to the correct peptides. From these samples, 16 unique proteins were assigned. For Agki venom, 133 high-quality MALDI–TOF-MS spectra were collected and 85 spectra matched the correct peptides. Agki venom had 15 unique confirmed proteins (Table 1; details in Supplementary Table II at

Following Sephadex G-50 GF, the pool of small venom proteins is expected to contain few, if any, large proteins (MM>10 kDa). Peptide identification of Naja venom by LC-MS/MS indicated that approx. 90% of proteins were cardio- and neuro-toxins, and over 91% had a MM less than 10 kDa. Protein degradation was apparent in the pool collected from late fractions of GF from the Agki sample, where approx. 90% of the proteins identified by MS have the mass values of more than 10 kDa. Surprisingly, the number of unique proteins verified by MALDI–TOF-MS from 2DE gel spots was much less than that of the identified proteins, with an average of six spots/unique protein. These data suggest that degradation or modification were prevalent in both samples. For instance, in Naja venom, 5′-END (5′-ectonucleotidase) from Rattus norvegicus (GI number 11024643) has theoretical pI and MM values of 6.5 and 64 kDa respectively. A total of 28 spots from 2DE were verified as 5′-END, with MMs ranging from 29 to 64 kDa and pIs ranging from 4.9 to 9.0. In Agki venom, a metalloproteinase from Gloydius halys (GI number 4106001) has theoretical pI and MM values of 5.3 and 70 kDa respectively; however, eight protein spots were identified as this enzyme on 2DE, with MMs ranging from 28 to 62 kDa and with pI values in the narrow range of 4.5 to 5.3. Interestingly, several venomous proteins were found to have higher MMs than their theoretical values. Haemorrhagic metalloproteinase kaouthiagin (GI number 32469675; 44 kDa) in Naja and salmobin (GI number 3668352; 29 kDa) in Agki migrated with MMs of 100 and 48 kDa respectively.

Summarized comparisons of snake venom proteomes
The overall comparison of venom proteins identified by the different approaches is summarized in Table 1. In total, 124 and 74 proteins were verified from Naja and Agki venoms respectively (see Supplementary Table III at Considering that current techniques limit the analysis of intact proteins by MS, generation of detectable peptides is crucial to the identification of proteins. In the present study, two different digestion approaches, shotgun and in-gel digestion, have been employed to achieve the peptide signals to a great extent. As described above, the samples from diluted snake venom or from the GF pool of small-mass proteins were chopped by shotgun tryptic digestion and subsequently analysed by LC-MS/MS. Combining the two approaches, a total of 110 and 56 proteins were identified from Naja and Agki respectively. With in-gel digestion after 1DE and 2DE, a total of 78 and 47 proteins were confirmed by MS from Naja and Agki respectively (see Supplementary Tables I and II at

As mentioned above, snake venom proteins are broadly categorized into three groups based on toxicity and two groups based on sequence homology. A general summary of the proteomes of Naja and Agki venom is briefly discussed below.

The haemotoxins are the major protein components in Agki venom, with a total of 26 confirmed proteins, including many involved in haemostatic regulation, such as serine proteases, coagulation factors, haemorrhagic toxins, disintegrin and fibrinogen clotting inhibitor (detailed in Supplementary Table III at In these identified haemotoxins, some belong to the same protein family, for instance, the sequence identities of Agki platelet aggregation disintegrins (GI numbers 104444, 118651, 25527065, 25527067, 25527068 and 25527069) are over 70%. Only three are haemotoxins found in Naja venom: two haemorrhagic metalloproteinases (GI numbers 20530119 and 32469675) and one salmobin (GI number 3044080). The haemorrhagic metalloproteinases are specific for the cobra species, and the salmobin is identical with one found in Agki venom.


Naja venom contains 11 confirmed neurotoxins, all of which are cobra-specific. Some Naja neurotoxins share high identity in amino acid sequences, such as cobrotoxin III (GI number 4185752) being 93% identical with cobrotoxin IV (GI number 4185750), and neurotoxin 6 (GI number 85982) sharing 71% identity with neurotoxin 7 (GI 85983). Only one neurotoxin, acutolysin from Deinagkistrodon acutus, was detected in Agki venom, which does not overlap any amino acid sequences from Naja neurotoxins.


As shown in Table 1, the cardiotoxins are the most abundant components of Naja venom, with MMs ranging from 6.7 to 10 kDa. Based upon the identities of amino acid sequences, the 68 Naja cardiotoxins could be broadly grouped into 14 different families, as illustrated in Supplementary Table III (available at Six cardiotoxins were verified in Agki venom, in which two cardiotoxins (GI numbers 1054811 and 3342766) are the same as Naja cardiotoxins, and the other four are viper-specific proteins.


Phospholipase A2
In contrast with other protein categories, phospholipase A2 is a common protein family in both snake venoms with relatively high abundance, represented by 15 and 13 proteins in Naja and Agki respectively. However, phospholipase A2 is species-dependent, and no common phospholipase A2 was identified in both species in this study. Of 15 Naja phospholipases A2, 67% of these proteins have over 70% identity in amino acid sequence; however, these have low similarity to Agki phospholipase A2.


In the present study, no metalloproteinase has been identified in Naja venom, even though two haemorrhagic metalloproteinases were found and categorized as haemotoxins. However, metalloproteinase was confirmed a major protein component in Agki venom using all of the proteomic methods. The Agki metalloproteinases usually have 30–60 kDa theoretical MMs and slightly acidic pI values, which are found in a good separation by 2DE. In Table 1(B), nevertheless, only five metalloproteinases were identified by 2DE-MALDI–TOF-MS; each of the other three approaches measured over ten metalloproteinases. Surprisingly, LC-MS/MS analysis monitored 14 metalloproteinases in the small MM pool (MM Taken together, the metalloproteinases were revealed to be strongly autocleaved either in the venom gland or during sample preparation.


Other proteins
This category represents, for the most part, species-specific proteins such as latisemin, ophanin and natrin specific for cobra, and saxatilin, catrocollastatin and mamushigin specific for viper. No common protein is shared by both snake venoms in this category.

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