Through this Presentation YOUNG RESEARCHERS could understand the sequencing of peptide molecules(de-novo sequencing) using tandem mass spectrometric data manually.

1. CiguatoxinConotoxin Snake toxin4 Spider toxin
Sequencing of Venompeptides
Rajesh. R P, Scientist, Sathyabama Institute of Science and Technology

2. Conus is a large genus of small to large predatory sea snails, marine
gastropod molluscs, with the common names of cone snails or cone shells
Conidae Currently contains over 800 recognized species cones.
Cone snails use a hypodermic-like modified radula tooth and a venom gland to
attack and paralyze their prey before engulfing it.
Cone snail venoms are mainly made of cocktail of peptides and proteins.
The venoms contain many different types of peptide toxins that vary in their effects;
some are extremely toxic.
The sting of small cones is no worse than a bee sting, but the sting of a few of the
larger species of tropical cone snails can be serious, occasionally even fatal to human
beings.

3. Conus tulipa
Conus victoriae
Conus ebraeus
FISH
MOLLUSC
WORMS
PISIVOROUS
MOLLUSCVOROUS
VERMIVOROUS

5. VENOM APPARATUS
VENOM BULB
VENOM DUCT

6. C. striatus, a Clade I species
(A) and C. tulipa, a Clade II
species (B).
1) C. striatus extends its
proboscis, harpoons the fish
and through a “lightning-
strike cabal” of toxins
causes an almost
instantaneous
immobilization of prey.
2) C. tulipa probably goes
after schools of fish hiding in
reefs at night using a net
strategy; once it has
engulfed the school with its
highly distensible rostrum, it
uses a “nirvana cabal of
toxins” to make the fish
quiescent for stinging them
and causing an irreversible
neuromuscular paralysis.
Conus Strategies for Catching Fish

7. Conotoxin Library- “CONOSERVER”
• Marine gastropods, better known as cone snails from Conus genus, produce a
mixture of venomous peptides for capturing prey, defense and deterring
competitors.
• These conopeptides can generally be divided into two broad classes, the
disulfide rich conotoxins which contain two or more disulfide bonds and the
disulfide poor which contain one or no disulfide bond.
• Conotoxins are small peptides ranging from 5-40 amino acids in length.
• Each cone snail may contain up to 200 peptides and given there are over 700
cone snail species, the library of bioactive peptides is huge (with a broad
estimate of thousands of unknown conopeptides .
• The targets for these toxins are a range of membrane bound receptors and ion
channels, thus exhibiting great potential as therapeutics themselves or as leads
to therapeutics.

8. B.M.Olivera et al ; current opinion in neurobiology 1999,9:772-777
Effects of different peptides from Conus geographus venom on mice
behavior

9. Applications of conotoxins
• Diagnosis of rare neuromuscular disorder
• Anticonvulsant, Anesthetic
• Pain killer (alternative to morphine)
• Basic bench research-(using toxins for neurophysiological studies).
• treatment of neurological diseases such as multiple sclerosis, shingles,
diabetic neuropathy and other painful neurological conditions.
• Many other target yet to be discovered as with unknown function many
conotoxins have been reported.
• Ziconotide (SNX-111; Prialt) is an atypical analgesic agent for the
amelioration of severe and chronic pain whic is Derived from Conus
magus

10. MAJOR GENE SUPERFAMILIES OF CONOTOXIN

11. • Conotoxins feature a vast array of post translational modifications (PTMs).
• PTMs are chemical or structural changes to the residues of a peptide that are not
encoded in the peptide gene.
• PTMs are introduced by specialized enzymes that change the nature of a specific
residue. It is believed that the enzymes involved identify their targets by their signal
and/or propeptide sequence.
POST TRANSLATIONAL MODIFICATIONS

12. De Novo Peptide Sequencing
"de novo peptide sequencing" is, peptide sequencing performed without prior knowledge
of the amino acid sequence.
Mass spectrometers do have the advantage when it comes to generating sequence data
for peptides in low femtomole quantities.
However, Edman degradation will always enjoy the advantage when pmol quantities of a
peptide are available.
At higher pmol quantities (2-10 pmol) and 99% purity, Edman will often provide the exact
amino acid sequence without ambiguity for a limited run of amino acids, 6-30 amino
acids. HIGH PURITY UP TO 99% required
MS/MS enjoys sensitivity, and speed, and does not require an external standard for each
amino acid or amino acid variant.
Even crude mixture could be analysed using Mass Spectrometry based denova
sequencing. LESS PURITY or CRUDE MAY WORK
Another advantage is that MS/MS sequencing is never stopped by a blocked amino
terminus, as is the case for Edman degradation.

13.  Peptide de novo sequencing is the analytical process that derives a peptide’s
amino acid sequence from its tandem mass spectrum (MS/MS) without the
assistance of a sequence database.
 It is in contrast to another popular peptide identification approach –
“database search”, which searches in a given database to find the target
peptide. A clear advantage of de novo sequencing is that it works for both
database and novel peptides.
Basic Principle
 In a tandem mass spectrometer, the peptide is fragmented along the peptide
backbone and the resulting fragment ions are measured to produce the
MS/MS spectrum.
 Depending on the fragmentation methods used, different fragment ion types
can be produced. The most widely used fragmentation methods today are
Collision-Induced Dissociation (CID) and Electron-Transfer Dissociation
(ETD). CID produces mostly b and y-ions; and ETD produces mostly c and
z-ions.

14. FRAGMENTATION OF PEPTIDES IN
MASS SPECTROMETRY

15. C C S Q D C R VC I O C C P Y
17
01
C C S Q D C R VC I O C C P Y
1701
A venom peptide with mass 1701(M+H) from vermivorous cone snail Conus figulinus

16. 6 Cysteines: 3-disulfide Bridge
756
AF New-16- TCEP-NEM
08-08-2011
REDUCTION USING TCEP(Tris-(2-Carboxyethyl)phosphine) AND
SUBSEQUENT ALKYLATION NEM(N-Ethylmaleimide )
C C S Q D C R VC I O C C P Y
C= NEM LABELLED
1702.8
0_F181: +MS
2209.1
2335.2
2458.4
0_H121: +MS
0
1
2
3
4
5
4x10
Intens.
0.0
0.2
0.4
0.6
0.8
1.0
4x10
1700 1800 1900 2000 2100 2200 2300 2400 2500 m/z

17. 2 Cysteines:
1disulfide Bridge
252
6 Cysteines:
3disulfide Bridge
756
4Cysteines: 2
disulfide Bridge
504
AF New-20- TCEP-NEM
08-08-2011
VGCODWQPWC
DCCWPGRPDCCAP
CONTRYPHAN
T SUPERFAMILY
M SUPERFAMILY
1187.7
1418.8
1528.4
1702.9
1804.2 2020.62077.12146.02215.5 2416.2 2519.0
0_F221: +MS
1260.5
1439.9
1535.3
1705.3
1799.2
1922.2
2049.4 2210.3
2335.4
2458.5
0_H141: +MS
0
1000
2000
3000
4000
Intens.
0
1000
2000
3000
4000
1200 1400 1600 1800 2000 2200 2400 m/z
Native Peptide
R & A Peptide

18. Lysine and Glutamine have nearly isobaric mass(128D), an acetylation of the
Lysine side chain differentiates these two amino acids by 42 u.
+

19. AFN 16(19-09- 2011)
AFN16(19-09-2011)
ACETYLATION
42
NH2-C C S Q D C R VC I O C C P Y
Reduced alkylated Peptide – Acetylated
with Acetic anhydride
2442.4
2460.4
2584.7
0_H121: +MS
2483.1
2502.1
2515.2
2529.3
2543.3
2557.4
0_C32: +MS
0.0
0.2
0.4
0.6
0.8
4x10
Intens.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
4x10
2440 2460 2480 2500 2520 2540 2560 m/z

20. AFN 16(14-09- 2011)
AFN16(14-09-2011)
ESTERIFICATION
2442.4
2460.4
0_H121: +MS
2416.1
2423.2
2432.1
2437.4
2442.2
2460.0
2468.0
2474.2
2488.2
2518.7
2523.5
0_A171: +MS
0
2000
4000
6000
8000
Intens.
0
500
1000
1500
2420 2440 2460 2480 2500 m/z
CCSQDCRVCIOCCPY-COOH
Reduced alkylated Peptide – Esterified
with Methanolic HCL

21. 2209.1
2335.2
2384.3
2460.4
2586.8
0_H121: +MS
842.4
1064.3
1189.4
1310.6
2383.9
0_A111: +MS
0
2000
4000
6000
8000
Intens.
0
1
2
3
4x10
800 1000 1200 1400 1600 1800 2000 2200 2400 m/z
AFN 16(14-09- 2011)
AFN16(14-09-2011)
TRYPSIN DIGESTION
C C S Q D C R VC I O C C P Y
Reduced alkylated Peptide – Digested
with trypsin at pH 7.5

22. 342.1
411.1
507.2
542.2
570.1
639.2
667.2
787.1
850.3
921.2
961.3
1011.8
1091.3
1139.9
1189.4
1221.4
1270.5
1339.5
1452.6
1498.5
1584.6
1614.5
1672.7
1953.7
2021.6
2181.7
175. +MS2(1230.3), 33.6-34.1min #2059-#2084
0
1
2
3
6x10
Intens.
400 600 800 1000 1200 1400 1600 1800 2000 m/z
457.0
b2
y3
b3
672.2
b4
735.2
y4
b5
1015.3
b6
848.3
y5
y6
y7
1288.6
y8
1171.4
b7
b8 b9
1611.6
1445.4y9
y10
1787.6
y11
2002.2
y13
1724.9
b11
b13
b10
b12
C C S Q D C R VC I O C C P Y
b1 b2 b3 b4 b5 b6 b7 b8 b9 b11b12b13 b14b10
y14 y13 y12y11 y10 y9 y8 y7y6 y4 y3 y2 y1y5
Hydroxylated Proline
DE-NOVO SQUENCING
OF FI1701

23. Contact Me:
Dr. R. P. Rajesh,Ph.D,
Scientist C,
Molecular & Nanomedical Sciences
Sathyabama Institute of Science and Technology, Chennai-119
Ph: 8072968170
+919994189582