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Structural Analysis Of Bioactive Peptides Biology Essay

Animal venom has many important pharmaceutical and agricultural properties. There are over 38 000 known spider species, however only a few have been described. It is known that Haplopelma lividum tarantula venom is fatal to mice within a short period of time, however every little is known about the structure of the venom. The aim of this study was to use mass spectrometry to analysis identify bioactive peptides in Haplopelma lividum tarantula venom. This was carried out on five fractions of the tarantula venom, matrix assisted laser desorption/ionization - time of flight (MALDI-TOF) mass spectrometry was used to determine the molecular weight. Reduction and alkylation and trypsin digests were also used to find out structural information about the peptides. The results showed that 2 of the fractions may contain novel peptides. There are also similarities between some of these peptides and peptide toxins from Haplopelma huwenum and Brachypelma albceps.

In the last 50 years animal venoms have become an important area of research, this is mainly due to the increased knowledge about the potential pharmaceutical and agricultural properties [1]. Even thought there are over 38 000 known species of spider, only a small proportion have been studied in detail, maybe as little as 60 species. It is known that the biological make up of spider venom is very complex, sometimes with up to 60 peptides present in the venom of one species [2].

Studies into peptide toxins have shown that the main targets are within the calcium, sodium and potassium ion channels. Other targets include the mechano-sensitive and acid-sensing ion channels (ASIC) [3]. While there are no drugs derived from spider peptide toxins at present, there are a number of areas of pharmaceutical interest, mainly cardiovascular and analgesic research.

Spider peptide toxins such as phrixotoxins (PaTx1 and PaTx2) and GsMTx4 both target different aspects of cardiac activity which means these toxins have huge potential in treatment of cardiovascular disease [4, 5, 6]. Sodium channels and ASIC have been identified as containing pain sensing neurons, which means treatment of pain with spider peptide toxins is a future possibility [4].

Analysis and sequencing of the peptide structure provide the best understanding of the pharmaceutical targets and venom mapping. This is initially carried out by a combination of HPLC, MALDI-TOF, Q-TOF and ion trap mass spectrometry. Further structural analysis is usually carried out using de novo sequencing [7].

This study aims to look at bioactive peptides from Haplopelma lividum tarantula venom. Not much is known about the H. lividum tarantula. What little information is available on this species comes from a review by Escoubas and Rash that show results from unpublished work by Escoubas. The results show the time in minutes for death to occur in mice after they have been injected with 0.1ml of crude tarantula venom, the venoms in the experiment are from tarantulas from different regions of the world [6]. Venom from the H. lividum tarantula was shown to kill the mice after only 10 minutes. This was similar to other tarantula species from Asia.

To investigate the bioactive peptides in the venom fractions, reduction and alkylation, trypsin digest and mass spectrometry were used.

2. Experimental/Methods and Materials

2.1 Materials

Five fractions (f 42, f 43, f 45, f 50 and f 57) of crude H. lividum tarantula venom where obtained from Spider Pharm, Yarnell, Texas, USA and had previously been fractionated by HPLC. DTT (Dithiotheriol) and 4-vinyl-pyridine were purchased from Sigma-Aldrich Ltd, Dorset, England, UK. Ammonium bicarbonate and a-cyano-4-hydroxycinnamic acid (CHCA) were purchased from Fluka, Devon, England, UK. Methanol and HPLC grade water were purchased from VWR International, Leciestershire, England, UK. TFA and ACN were purchased from Applied Biosystems Inc, Foster City, California, USA. Trypsin was purchased from Hampshire, England, UK.

2.2 Reduction and Alkylation

To identify the presence of cysteines 50ml of raw venom extract mixed with 50ml of 100mM DTT, 500ml of 20mM ammonium bicarbonate and 10ml of 4-vinyl-pyridine. The sample was wrapped in foil and placed in a water bath (37oC) for 90 minutes. The samples were then spun in the centrifuge for 10 minutes to remove the 4-vinyl-pyridine from the mixture.

2.3 Trypsin Digest

To determine if trypsin was present. 100ml of trypsin was added to the vinyl-pyridine samples of f 42 and f 45. The mixture was wrapped in foil and placed into a water bath (37oC) for 1 hour.

2.4 ZipTip

Tips were prepared using cycles of aspirations (to wet the tip) and dispensing to get rid of the waste, below is the method used:

The tip was initially activated by 10 cycles of methanol followed by 10 cycles of 0.1% TFA, then 50 cycles of the venom fraction, then another 5 cycles of 0.1% TFA (each aspiration was discarded in to a separate tube). Then 80% ACN, 19.9% water and 0.1% TFA was added to the tip and 1.5µl was added to the MALDI-TOF MS sample plate.

2.5 MALDI-TOF Mass Spectrometry

The Voyager-DE Biospectrometry Workstation from Applied Biosystems, Foster City, California, USA, was used for MALDI-TOF MS analysis, data was gathered with Applied Biosystems software and exported using Data Explorer. 1ml of sample is placed on a well on the MALDI-TOF sample plate, 1ml of CHCA is added to the well. The mixture was left to dry, then the plate was inserted into the MALDI-TOF mass spectrometer, and the molecular weight was read.

2.6 Q-TOF and Ion Trap Mass Spectrometry

The Micromass Q-ToF Ultima API from Micromass UK Ltd, Manchester, England, UK was used along with Masslynx and PepSeq software for analysis. Ion trap MS was carried out on the LCQ electrospray ion trap mass spectrometer from Thermo Finnigan, San Jose, California, USA. XCalibur software from Thermo Finnigan was used to monitor and control the system.

3. Results

3.1 MALDI-TOF MS and Reduction and Alkylation

Each fraction was run on the MALDI-TOF MS to determine the original molecular weight of the crude venom (Table 1). Research carried out on the molecular weights of the 5 fractions showed similarities with the Haplopelma huwenum and Brachypelma albiceps tarantulas [8]. Using arachnoserver (http://www.arachnoserver.org) close matches for three of the fraction were identified. After the reduction and alkylation was carried out, the ZipTips where used to improve the quality of the data (Table 1). This showed that cysteines were present in two fractions and absent in another two. However, this method of reduction and alkylation seems to have unexplainably removed f 57. These results where used to determine which samples would be used for further analysis.

3.2 Trypsin Digest

The next step was to carry out a trypsin digest. It was decided f 42 and f 45 would be used to determine if there are any differences caused by the presence of cysteines.

When the reduction and alkylation was carried out on f 42 there was an increase in molecular weight suggesting cysteines presences, there was also an obvious decrease in the intensity of the peak (Figure 1 A and B). When the trypsin digest sample was run on the MALDI-TOF MS, no significant peaks were produced (Figure 1C).

It was clear that no significant change occurred as a result of the reduction and alkylation of f 45 (Figure 1 D and E). When the trypsin digest was carried out two clear peaks, at smaller molecular weights, could be seen (Figure 1 F).

3.3 Q-TOF and Ion Trap Mass Spectrometry

The trypsin digest sample of f 45 was then run on the Q-TOF MS, however no positive results were obtained from the sample.

F 43 was also investigated further. It was clear from the reduction and alkylation that cysteines were present in this fraction (Figure 2 A and B). The next step was to run the crude, unmodified fraction on the ion trap MS (Figure 3 A and B). F 43 was chosen for further analysis as it had the smallest original molecular weight. The fraction was run on the ion trap MS and compared to a database of known arachnid sequences (Figure 3 A), a number of sequences were found with similar molecular weights. A BLAST search (http://blast.ncbi.nlm.nih.gov/) was run and most of the sequences came back to Ixodes scapularis (deer tick) (Table 2). This fraction was also run on the Q-TOF MS however there were no positive results.

A

4. Discussion

Spider peptides are complex molecules that vary in size and structure. This is evident from the results collected, with original molecular weights ranging from 3707.33-6725.00 Da in one spider species.

The results showed that f 42 had a molecular weight of 4289.09 Da and further investigation suggested the presence of 6 cysteines. Using arachnoserver a peptide toxin from H. huwenum known as U1-theraphotoxin Hh1a was found with a molecular weight of 4281.02 Da. It was also shown to have 6 cysteines and experimental data suggesting the presence of disulfide bonds [9, 10]. This toxin was discovered in 1998, as of yet the target is unknown. It is known to be fatal to mice after intracerebroventricular injection, it can also reversibly paralyze both cockroaches and locusts.

F 45, which had a molecular weight of 4429.50 Da, has two possible matches, U10-theraphotoxin Hh1a from H. huwenum and U1-theraphotoxin Ba1b from B. albiceps. Both peptide toxins have cysteines present, which is not the case for f 45. U10-theraphotoxin was discovered in 2008 and as of yet both the target and function are unknown. U1-theraphotoxin Ba1b was also discovered in 2008 and the target is still unknown. However, toxicity experiments have shown that it is fatal to crickets. No unpleasant effects were observed in mice when given at a dose of 1µg/g intracranially or into the body cavity.

F 57 was the largest of the 5 fraction with a molecular weight of 6725.00 Da, however the reduction and alkylation carried out did not yield a positive results. The arachnoserver found a peptide toxin, U7-theraphotoxin Hh3a from the H. huwenum spider with a molecular weight of 6737.96. This peptide toxin was discovered in 2008 and the target and function are still unknown. There were also cysteines present in the sequence. As the reduction and alkylation of f 57 did not produce results a clear comparison can not be made. It is possible that due to the size of the fraction, a different reduction and alkylation method may be needed, this may allow further investigation of the fraction.

From the results obtained, it is clear to see that f 42 is very similar to U1-theraphotoxin Hh1a, it may also act in the same way, however more information would be needed. It is plausible to suggest that f 43 and f 45 may be novel peptides, with further investigation, this may also be possible for f 50 and f 57. This would include further sequencing with methods such as edman degradation.

The majority of analysis was carried out on the MALDI-TOF MS. While these results where informative, with regards cysteine and trypsin presence, they were limited. Ion trap MS was used on one fraction and was able to provide more sequencing information however the suggested sequences were from unrelated genus of spider (Table 2). Q-TOF MS was used but this did not provide any positive results.

Corzo et al have suggested that there may be a relationship between H. huwenum and B. albiceps due to recent sequencing information that has been obtained [11]. As there is very little information on H. lividum at present, and the results from arachnoserver suggest similarities with both H. huwenum and B. albiceps, it may be possible with further analysis to include H. lividum in the relationship that Corzo et al has discussed.

5. Conclusion

The results have shown that two of the fractions (f 43 and f 45) may be novel peptides. F 50 and f 57 require further analysis, however there is potential for four novel peptides to come from this study. The next step would be to carry out edman degradation to identify the peptide sequences. After this identification of the peptide targets could be carried out.

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