Cystine, an Essential Determinant of Protein Tertiary ...tools.thermofisher.com/content/sfs/posters/PN-71010-AOAC2014-Cystine... · Cystine, An Essential Determinant Of Protein Tertiary

Cystine, an Essential Determinant of Protein Tertiary Structure, Is Also a Target for Electrochemical ManipulationQi Zhang, Bruce Bailey, Marc Plante and Ian Acworth Thermo Fisher Scientific, Chelmsford, MA, USA

2 Cystine, an Essential Determinant of Protein Tertiary Structure, Is Also a Target for Electrochemical Manipulation

Cystine, An Essential Determinant Of Protein Tertiary Structure, Is Also A Target For Electrochemical ManipulationQi Zhang, Bruce Bailey, Marc Plante and Ian AcworthThermo Fisher Scientific, Chelmsford, MA, USA

OverviewPurpose: Development of UHPLC and HPLC-electrochemical (EC) detection methods for the measurement of different cystine-containing peptides including simple peptides ( l t thi ) di i d tid (i li ) li tid ( t i d

Results

An instrumental prerequisite for trace analysis is that the HPLC system must be

2) Analysis Of Oxytocin, Vasopressin and InsulinMethodsHPLC System: Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC system including:

HPG 3600RS

The ability to measure more complex cystine-containing peptides and protiens using HPLC with electrochemical detection on a BDD working electrode was then evaluated. Two cyclic peptide hormones formed by a cystine bridge (oxytocin and vasopressin),

d l t i h h i b th i t d i t h i ti

Cyclotides are small disulfide rich peptides containing 28-37 amino acid residues and are isolated from plants. The tertiary structures of these peptides are very interesting as the three cystine bridges form a cystine knot motif (Figure 1) imparting great stability to the peptide. Today, over 100 cyclotides

1) Analysis Of Glutathione Redox Status

3) Analysis of Cyclotide by HPLC-EC detection

(glutathione), medium sized peptides (insulin), cyclic peptides (oxytocin and vasopressin) and peptides with cystine knots (cyclotides).

Methods: UHPLC- and HPLC-based methods using electrochemical detection with a robust boron-doped diamond (BDD) working electrode are described.

Results: The novel BDD working electrode extends the range of analytes capable of

An instrumental prerequisite for trace analysis is that the HPLC system must be inert (free from leachable transition metals) in order to achieve optimal sensitivity using an electrochemical detector. The system uses biocompatible materials in the flow path to reduce the influence of metal that contribute to elevated background currents at the electrochemical cell. Use of the Thermo Scientific Dionex 6041RS

lt A l ti l C ll id th i l t h i l biliti f th b

• HPG-3600RS pump • WPS-3000TRS autosampler• 3000 TCC-3000RS column thermostat• DAD-3000RS diode array detector • ECD-3000RS electrochemical detector with 6041RS ultra Analytical Cell and boron doped diamond (BBD) electrode

and a more complex protein hormone having both inter- and intra-chain cystinelinkages (insulin), were chosen. Chromatographic separation1 of all three compounds on a phenyl hexyl column is presented in Figure 3. Figure 4 shows HDV of the three peptides. The estimated limit of detection was 100-200 pg on column for both vasopressin and oxytocin. The ability to measure GSH and GSSG (above) shows that oxidation of both thiols and disulfides can contribute to the overall electrochemical

( g ) p g g y p p y, yhave been isolated and characterized from a number of species including the Rubiaceae, Violaceae, and Cucurbitaceae. In the plant cyclotides act as both natural insecticides and anti-microbial agents. The cyclotide structure is of interest to pharmaceutical companies for the development of potential anti-HIV and anti-tumor agents. For these studies, the cyclotide cycloviolacin O2, a 30

Results: The novel BDD working electrode extends the range of analytes capable of being measured by EC detection, making measurement of low levels of peptides possible.

Introduction

ultra Analytical Cell provides the unique electrochemical capabilities of the boron-doped diamond which enables the oxidation of organic compounds using higher electrode potentials than other working electrode materials.

The direct electrochemical detection of aminothiol compounds only using a boron-Column: Thermo Scientific™ Accucore™ RP MS column

boron doped diamond (BBD) electrode oxidation of both thiols and disulfides can contribute to the overall electrochemical signal obtained from these peptides. In the case of oxytoxin and vasopressin the contribution from the oxidation of cystine maybe augmented by other residues that can undergo oxidation (e.g., each contains a tyrosine residue). The electrochemical behavior of insulin is far more complex. Contribution to the overall response will come from oxidation of the three cystine residues but each polypeptide chain has two

residue cyclic peptide found in the sweet violet (Viola odorata) was studied2,3. Figure 5 shows the detection of cycloviolacin O2 both as the standard and its presence in a crude extract of the sweet violet.

Figure 5. Chromatogram showing detection of cycloviolacin O2 and it’s presence in the crude extract of sweet violet using BDD cell

1) Analysis Of Glutathione Redox Status by UHPLC-EC Detection

The ability for thiol residues to undergo oxidation to disulfides is biochemically important , since it affects antioxidant activity (e.g., glutathione) and determiniation of protein tertiary structure. The formation of cystine disulfide bridges from two cysteineresidues not only influences protein confirmation but also renders it electrochemically active. Although peptides can be quantified by electrochemical oxidation of the disulfide

doped diamond electrochemical cell has been previously described.1,2 The applied potential used for this study (+1600 mV) was sufficient to oxidize both thiol and disulfide analytes. Advantages of this approach include a stable electrode surface and method simplicity since no sample derivatization is required. After each analysis the electrode surface was regenerated by a 10 second clean cell pulse at

Column: Thermo Scientific Accucore RP-MS column ,2.6 μm, 2.1 150 mm

Pump Flow Rate: 0.500 mL/minMobile Phase: 0.1% pentafluoropropionic acid, 0.02% ammonium

hydroxide, 2.5% acetonitrile, 97.4% water

from oxidation of the three cystine residues, but each polypeptide chain has two tyrosine residues that can also respond. Although insulin is a small protein (51 residues) its ability to oxidize will be dependent upon interaction between its resides and the BDD working electrode surface. Kinetic hindrance will result in a decreased signal (proportional to 1/MW).

presence in the crude extract of sweet violet using BDD cell.

bond, the commonly used working electrode, glassy carbon, suffers from two major issues – a limited oxidation potential and the persistent fouling of the electrode’s surface necessitating frequent cleaning procedures. The boron-doped diamond (BDD) working electrode, on the other hand, can be used at extreme oxidation potentials and, as its surface is inert, does not suffer from adsorption problems. The ability for HPLC

f f f

y g y p+1900 mV. After a short re-equilibration period the electrode was once again stable and could be used for the analysis of the next sample. Figure 2 illustrates the overlay of calibration standards for these compounds ranging from 1–20 µg/mL.Excellent peak resolution and retention time uniformity were observed. The column used for this method was the Accucore RP-MS 2.6 micron solid-core

yColumn Temperature: 50.0 CPost Column Temperature 25.0 CInjection Volume: 2 µL standards; 4 µL samplesCell Potential: 1600 mVFilt C t t 1 0

Figure 3. Chromatogram showing detection of 8-vasopression, oxytocinand insulin electrochemically with boron doped diamond electrode at 1800mV.

010914-vasopressin #119 3 mix 10ug/mL phenyl-hexy TFA-LiClO4 1800mV ECDRS 1 ent [

µA]

Crude extract of sweet violet, 5 mg/mLwith electrochemical detection to quantify a variety of disulfide containing peptides was evaluated and included measurement of GSH and glutathione disulfide (GSSG), cyclic nonapeptides (vasopressin and oxytocin), an example of intra and inter chain disulfide cross-linking (insulin) and a cyclic, disulfide rich cyclotide (cycloviolacin O2) (Figure 1).

column used for this method was the Accucore RP MS 2.6 micron solid core material which provides fast, high resolution separations but with lower system pressures.

Figure 2. Overlay of GSH, GSSG and related analytical standards ranging from 1–20 µg/mL (CysGly – cysteinyl-glycine; HCYS – homocysteine).

Filter Constant: 1.0 sCell Clean: OnCell Clean Potential: 1900 mVCell Clean Duration: 10.0 s 3,000

4,000

V i

Oxytoxin nA

010914 vasopressin #119 3 mix 10ug/mL phenyl hexy TFA LiClO4 1800mV ECDRS_1

Cur

re

g

Cycloviolacin O2, 100 µg/mL

C l i

Figure 1. Structures of peptides used during method development.

g g µg ( y y y y g y ; y )

2.2

2.4

2.6

2.8

3.0 µA

ysG

ly

GS

H

Met

hion

ine

Column: Accucore phenyl hexyl column, 2 6 2 1 100 1 000

2,000

Cur

rent

[nA

]

Vasopressin

Insulin

2) Analysis Of Oxytocin, Vasopressin and Insulin by HPLC-EC Detection

Conclusion• UHPLC/HPLC separation and detection on a BDD working electrode is a practical

approach to the measurement of cystine-containing peptides and proteins.• The BDD working electrode overcomes the practical limitations of traditional carbon

working electrodes. 1 2

1.4

1.6

1.8

2.0

2.2

Cy

HC

YS

GS

SG

2.6 μm, 2.1 100 mm Pump Flow Rate: 0.600 mL/minMobile Phase: A: 50mM lithium perchlorate, 0.05% TFA in water

B: 50mM lithium perchlorate, 0.05% TFA in acetonitrileGradient: 15 to 50% B in 10min 500

0

1,000

min g• Future work will a) evaluate the contribution of other amino acid residues to theoverall response of the peptide/protein and b) the inclusion of a BDD workingelectrode prior to the electrospray source of a mass spectrometer to study themechanism of peptide/protein oxidation and possible cleavage reactions.4

B) GSSG (oxidized form)A) GSH (reduced form)

0.4

0.6

0.8

1.0

1.2

2 µg/mL

5 µg/mL

10 µg/mL

20 µg/mL Gradient:Column Temperature:

15 to 50% B in 10min35.0 C

Injection volume: 5 µLCell Potential: 1800 mVFilter Constant: 1.0 s

0.00 1.25 2.50 3.75 5.00 6.25 7.50 8.75 10.00 11.00Time [min]

-500

AcknowledgementsThe standard of cycloviolacin O2 and the crude extract of the sweet violet were kindly provided by Professor Ulf Göransson from Uppsala University in Sweden.0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0

-0.0

0.21 µg/mL

min

G f ff

Filter Constant: 1.0 sCell Clean: OnCell Clean Potential: 1950 mVCell Clean Duration: 20.0 s

Figure 4. HDV of vasopressin, oxytocin and insulin on a BDD electrode.

400HDV of Vasopressin, Oxytocin and Insulin

References 1. Suresh Kumar, K.; Kavitha, M.P.; Dharuman, J.; and Dhandapani, B. RP-HPLC

method development and validation for the estomation of oxytocin in milk. Inl. J. of ChemTech Res. 2010, 2, 1340-1343.

Oxytocin Vasopressin

Good linearity of response to different concentrations was obtained with correlation coefficients ranging from R2 = 0.989–1.00 for the five compounds evaluated (Table 1) over the range of 1–20 µg/mL. The percent relative standard deviation (%RSD) for calibration curves (five concentrations in triplicate) is shown.

Column: Thermo Scientific™ Acclaim™ C18 2.2 μm, 3 x 75mm 300

350

400

n)

VasopressinOxytocinInsulin

3) Analysis Of Cyclotides by HPLC-EC Detection

, ,

2. Herrmann, A.; Burman, R.; Mylne, J.S.; karlsson, G.; Gullbo, J.; Craik, D.J.; Clark, R.J.; Göransson, U. The alphine violet, Viola biflora, is a rich source of cyclotides with potent cyctotoxcity. Phytochemistry. 2008, 69, 939-52.

3. Ovesen, R.G.; Göransson, U.; Hansen, S.H.; Nielsen, J.; Hansen, H.C. A liquid

Table 1. Regression data for standard calibration curves.

Peak #Points

RSD%

CorrelationCoefficient Slope

μ ,Pump Flow Rate: 0.500 mL/minMobile Phase: A: 50mM ammonium formate in water, pH4.4

B: 65% acetonitrile, 15% water, 20% 100mM ammonium formate at pH4.4

200

250

Respon

se (n

A*min

chromatography-electrospray ionization-mass spectrometry method for quantification of cyclotides in plants avoiding sorption during sample preparation. J. Chromatogr A. 2011, 1218, 7964-70.

4. Permentier, H.P., Bruins, A.P. Electrochemical oxidation and cleavage of proteins with on-line mass spectrometric detection J Am Soc Mass Spectrom 2004

Cyclotide

CysGly 15 8.9059 0.989 0.0481GSH 15 1.7446 1.00 0.0509Meth 15 3.4526 0.999 0.0736

HCYS 15 1.9282 1.00 0.0268

Column Temperature: 45.0 CGradient: 0-15min, 30-100%B,; 15-20min, 100%BInjection Volume: 10 µL standardsCell Potential: 1500 mV 50

100

150

Area

R

Human Insulinwith on line mass spectrometric detection. J. Am. Soc. Mass Spectrom. 2004, 15, 1707-1716.GSSG 15 1.2458 1.00 0.0170Cell Clean Potential: 1900 mV

Cell Clean Duration: 10.0 s 0400 600 800 1000 1200 1400 1600 1800 2000

Applied potentail (mV)PO71010_E 03/14S

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

3Thermo Scientific Poster Note • PN71010_PITTCON_2014_E_03/14S



Results


2) Analysis Of Oxytocin, Vasopressin and InsulinMethodsHPLC System: Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC system including:

HPG 3600RS






(glutathione), medium sized peptides (insulin), cyclic peptides (oxytocin andvasopressin) and peptides with cystine knots (cyclotides).



An instrumental prerequisite for trace analysis is that the HPLC system must beinert (free from leachable transition metals) in order to achieve optimal sensitivityusing an electrochemical detector. The system uses biocompatible materials in the flow path to reduce the influence of metal that contribute to elevated background currents at the electrochemical cell. Use of the Thermo Scientific Dionex 6041RS


• HPG-3600RS pump • WPS-3000TRS autosampler• 3000 TCC-3000RS column thermostat• DAD-3000RS diode array detector• ECD-3000RS electrochemical detector with 6041RS ultra Analytical Cell andboron doped diamond (BBD) electrode


( g ) p g g y p p y, yhave been isolated and characterized from a number of species including the Rubiaceae, Violaceae, and Cucurbitaceae. In the plant cyclotides act as both natural insecticides and anti-microbial agents. The cyclotide structure is of interest to pharmaceutical companies for the development of potential anti-HIVand anti-tumor agents. For these studies, the cyclotide cycloviolacin O2, a 30

Results: The novel BDD working electrode extends the range of analytes capable ofbeing measured by EC detection, making measurement of low levels of peptides possible.

Introduction



boron doped diamond (BBD) electrode oxidation of both thiols and disulfides can contribute to the overall electrochemicalsignal obtained from these peptides. In the case of oxytoxin and vasopressin the contribution from the oxidation of cystine maybe augmented by other residues that can undergo oxidation (e.g., each contains a tyrosine residue). The electrochemical behavior of insulin is far more complex. Contribution to the overall response will comefrom oxidation of the three cystine residues but each polypeptide chain has two

residue cyclic peptide found in the sweet violet (Viola odorata) was studied2,3.Figure 5 shows the detection of cycloviolacin O2 both as the standard and its presence in a crude extract of the sweet violet.

Figure 5. Chromatogram showing detection of cycloviolacin O2 and it’spresence in the crude extract of sweet violet using BDD cell


The ability for thiol residues to undergo oxidation to disulfides is biochemicallyimportant , since it affects antioxidant activity (e.g., glutathione) and determiniation ofprotein tertiary structure. The formation of cystine disulfide bridges from two cysteineresidues not only influences protein confirmation but also renders it electrochemicallyactive. Although peptides can be quantified by electrochemical oxidation of the disulfide

doped diamond electrochemical cell has been previously described.1,2 The applied potential used for this study (+1600 mV) was sufficient to oxidize both thiol and disulfide analytes. Advantages of this approach include a stable electrode surface and method simplicity since no sample derivatization is required. After eachanalysis the electrode surface was regenerated by a 10 second clean cell pulse at




from oxidation of the three cystine residues, but each polypeptide chain has twotyrosine residues that can also respond. Although insulin is a small protein (51 residues) its ability to oxidize will be dependent upon interaction between its resides and the BDD working electrode surface. Kinetic hindrance will result in a decreased signal (proportional to 1/MW).


bond, the commonly used working electrode, glassy carbon, suffers from two major issues – a limited oxidation potential and the persistent fouling of the electrode’s surface necessitating frequent cleaning procedures. The boron-doped diamond (BDD)working electrode, on the other hand, can be used at extreme oxidation potentials and, as its surface is inert, does not suffer from adsorption problems. The ability for HPLC

f f f





µA]


column used for this method was the Accucore RP MS 2.6 micron solid corematerial which provides fast, high resolution separations but with lower system pressures.



4,000

V i

OxytoxinnA


Cur

re

g


C l i



2.2

2.4

2.6

2.8

3.0 µA

ysG

ly

GS

H

Met

hion

ine

Column: Accucore phenyl hexyl column,2 6 2 1 100 1 000

2,000

Cur

rent

[nA

]

Vasopressin

Insulin





1.4

1.6

1.8

2.0

2.2

Cy

HC

YS

GS

SG

2.6 μm, 2.1 100 mm Pump Flow Rate: 0.600 mL/minMobile Phase: A: 50mM lithium perchlorate, 0.05% TFA in water


0

1,000



0.4

0.6

0.8

1.0

1.2

2 µg/mL

5 µg/mL

10 µg/mL

20 µg/mLGradient:Column Temperature:

15 to 50% B in 10min35.0 C


0.00 1.25 2.50 3.75 5.00 6.25 7.50 8.75 10.00 11.00Time [min]

-500

AcknowledgementsThe standard of cycloviolacin O2 and the crude extract of the sweet violet were kindlyprovided by Professor Ulf Göransson from Uppsala University in Sweden.0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0

-0.0

0.21 µg/mL

min

G f ff




References1. Suresh Kumar, K.; Kavitha, M.P.; Dharuman, J.; and Dhandapani, B. RP-HPLC




Column: Thermo Scientific™ Acclaim™ C18 2.2 μm, 3 x 75mm 300

350

400

n)



, ,




Peak #Points

RSD%



B: 65% acetonitrile, 15% water, 20% 100mM ammonium formate at pH4.4

200

250

Respon

se(nA*

min



Cyclotide

CysGly 15 8.9059 0.989 0.0481GSH 15 1.7446 1.00 0.0509Meth 15 3.4526 0.999 0.0736

HCYS 15 1.9282 1.00 0.0268


100

150

Area

R

Human Insulinwith on line mass spectrometric detection. J. Am. Soc. Mass Spectrom. 2004,15, 1707-1716.GSSG 15 1.2458 1.00 0.0170Cell Clean Potential: 1900 mV


Appliedpotentail (mV)PO71010_E 03/14S





Results


2) Analysis Of Oxytocin, Vasopressin and InsulinMethodsHPLC System: Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC systemincluding:

HPG 3600RS









An instrumental prerequisite for trace analysis is that the HPLC system must be inert (free from leachable transition metals) in order to achieve optimal sensitivity using an electrochemical detector. The system uses biocompatible materials in the flow path to reduce the influence of metal that contribute to elevated background currents at the electrochemical cell. Use of the Thermo Scientific Dionex 6041RS


• HPG-3600RS pump• WPS-3000TRS autosampler• 3000 TCC-3000RS column thermostat• DAD-3000RS diode array detector • ECD-3000RS electrochemical detector with 6041RS ultra Analytical Cell and boron doped diamond (BBD) electrode




Introduction








doped diamond electrochemical cell has been previously described.1,2 The applied potential used for this study (+1600 mV) was sufficient to oxidize both thiol and disulfide analytes. Advantages of this approach include a stable electrode surface and method simplicity since no sample derivatization is required. After each analysis the electrode surface was regenerated by a 10 second clean cell pulse at







f f f

y g y p+1900 mV. After a short re-equilibration period the electrode was once again stableand could be used for the analysis of the next sample. Figure 2 illustrates the overlay of calibration standards for these compounds ranging from 1–20 µg/mL.Excellent peak resolution and retention time uniformity were observed. The column used for this method was the Accucore RP-MS 2.6 micron solid-core




µA]


column used for this method was the Accucore RP MS 2.6 micron solid core material which provides fast, high resolution separations but with lower system pressures.



4,000

V i

OxytoxinnA


Cur

re

g


C l i



2.2

2.4

2.6

2.8

3.0 µA

ysG

ly

GS

H

Met

hion

ine


2,000

Cur

rent

[nA

]

Vasopressin

Insulin





1.4

1.6

1.8

2.0

2.2

Cy

HC

YS

GS

SG

2.6 μm, 2.1 100 mmPump Flow Rate: 0.600 mL/minMobile Phase: A: 50mM lithium perchlorate, 0.05% TFA in water


0

1,000



0.4

0.6

0.8

1.0

1.2

2 µg/mL

5 µg/mL

10 µg/mL

20 µg/mL Gradient:Column Temperature:

15 to 50% B in 10min35.0 C


0.00 1.25 2.50 3.75 5.00 6.25 7.50 8.75 10.00 11.00Time [min]

-500


-0.0

0.21 µg/mL

min

G f ff








Column: Thermo Scientific™ Acclaim™ C18 2.2 μm, 3 x 75mm300

350

400n)



, ,




Peak #Points

RSD%



B: 65% acetonitrile, 15% water, 20% 100mMammonium formate at pH4.4

200

250

Respon

se(nA*

min



Cyclotide

CysGly 15 8.9059 0.989 0.0481GSH 15 1.7446 1.00 0.0509Meth 15 3.4526 0.999 0.0736

HCYS 15 1.9282 1.00 0.0268


100

150

Area

R





5Thermo Scientific Poster Note • PN71010_PITTCON_2014_E_03/14S



Results



HPG 3600RS















Introduction



boron doped diamond (BBD) electrode oxidation of both thiols and disulfides can contribute to the overall electrochemical signal obtained from these peptides. In the case of oxytoxin and vasopressin the contribution from the oxidation of cystine maybe augmented by other residues that can undergo oxidation (e.g., each contains a tyrosine residue). The electrochemical behavior of insulin is far more complex. Contribution to the overall response will come from oxidation of the three cystine residues but each polypeptide chain has two









from oxidation of the three cystine residues, but each polypeptide chain has two tyrosine residues that can also respond. Although insulin is a small protein (51 residues) its ability to oxidize will be dependent upon interaction between its resides and the BDD working electrode surface. Kinetic hindrance will result in a decreased signal (proportional to 1/MW).



f f f





µA]





4,000

V i

Oxytoxin nA


Cur

re

g


C l i



2.2

2.4

2.6

2.8

3.0 µA

ysG

ly

GS

H

Met

hion

ine


2,000

Cur

rent

[nA

]

Vasopressin

Insulin





1.4

1.6

1.8

2.0

2.2

Cy

HC

YS

GS

SG



0

1,000



0.4

0.6

0.8

1.0

1.2

2 µg/mL

5 µg/mL

10 µg/mL


15 to 50% B in 10min35.0 C


0.00 1.25 2.50 3.75 5.00 6.25 7.50 8.75 10.00 11.00Time [min]

-500


-0.0

0.21 µg/mL

min

G f ff









350

400

n)



, ,




Peak #Points

RSD%




200

250

Respon

se (n

A*min



Cyclotide

CysGly 15 8.9059 0.989 0.0481GSH 15 1.7446 1.00 0.0509Meth 15 3.4526 0.999 0.0736

HCYS 15 1.9282 1.00 0.0268


100

150

Area

R



Applied potentail (mV)PO71010_E 03/14S





Results



HPG 3600RS













( g ) p g g y p p y, yhave been isolated and characterized from a number of species including the Rubiaceae, Violaceae, and Cucurbitaceae. In the plant cyclotides act as both natural insecticides and anti-microbial agents. The cyclotide structure is of interest to pharmaceutical companies for the development of potential anti-HIV and anti-tumor agents. For these studies, the cyclotide cycloviolacin O2, a 30


Introduction




residue cyclic peptide found in the sweet violet (Viola odorata) was studied2,3. Figure 5 shows the detection of cycloviolacin O2 both as the standard and its presence in a crude extract of the sweet violet.

Figure 5. Chromatogram showing detection of cycloviolacin O2 and it’s presence in the crude extract of sweet violet using BDD cell










f f f





µA]





4,000

V i

OxytoxinnA


Cur

re

g


C l i



2.2

2.4

2.6

2.8

3.0 µA

ysG

ly

GS

H

Met

hion

ine


2,000

Cur

rent

[nA

]

Vasopressin

Insulin





1.4

1.6

1.8

2.0

2.2

Cy

HC

YS

GS

SG



0

1,000



0.4

0.6

0.8

1.0

1.2

2 µg/mL

5 µg/mL

10 µg/mL


15 to 50% B in 10min35.0 C


0.00 1.25 2.50 3.75 5.00 6.25 7.50 8.75 10.00 11.00Time [min]

-500

AcknowledgementsThe standard of cycloviolacin O2 and the crude extract of the sweet violet were kindly provided by Professor Ulf Göransson from Uppsala University in Sweden.0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0

-0.0

0.21 µg/mL

min

G f ff




References 1. Suresh Kumar, K.; Kavitha, M.P.; Dharuman, J.; and Dhandapani, B. RP-HPLC

method development and validation for the estomation of oxytocin in milk. Inl. J.of ChemTech Res. 2010, 2, 1340-1343.




350

400

n)



, ,

2. Herrmann, A.; Burman, R.; Mylne, J.S.; karlsson, G.; Gullbo, J.; Craik, D.J.;Clark, R.J.; Göransson, U. The alphine violet, Viola biflora, is a rich source ofcyclotides with potent cyctotoxcity. Phytochemistry. 2008, 69, 939-52.



Peak #Points

RSD%




200

250

Respon

se(nA*

min

chromatography-electrospray ionization-mass spectrometry method forquantification of cyclotides in plants avoiding sorption during sample preparation.J. Chromatogr A. 2011, 1218, 7964-70.

4. Permentier, H.P., Bruins, A.P. Electrochemical oxidation and cleavage of proteinswith on-line mass spectrometric detection J Am Soc Mass Spectrom 2004

Cyclotide

CysGly 15 8.9059 0.989 0.0481GSH 15 1.7446 1.00 0.0509Meth 15 3.4526 0.999 0.0736

HCYS 15 1.9282 1.00 0.0268


100

150

Area

R

Human Insulinwith on line mass spectrometric detection. J. Am. Soc. Mass Spectrom. 2004, 15, 1707-1716.GSSG 15 1.2458 1.00 0.0170Cell Clean Potential: 1900 mV




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Africa +43 1 333 50 34 0Australia +61 3 9757 4300Austria +43 810 282 206Belgium +32 53 73 42 41Brazil +55 11 3731 5140Canada +1 800 530 8447China 800 810 5118 (free call domestic)

400 650 5118

Denmark +45 70 23 62 60Europe-Other +43 1 333 50 34 0Finland +358 9 3291 0200France +33 1 60 92 48 00Germany +49 6103 408 1014India +91 22 6742 9494Italy +39 02 950 591

Japan +81 6 6885 1213Korea +82 2 3420 8600Latin America +1 561 688 8700Middle East +43 1 333 50 34 0Netherlands +31 76 579 55 55 New Zealand +64 9 980 6700 Norway +46 8 556 468 00

Russia/CIS +43 1 333 50 34 0Singapore +65 6289 1190Sweden +46 8 556 468 00 Switzerland +41 61 716 77 00Taiwan +886 2 8751 6655UK/Ireland +44 1442 233555USA +1 800 532 4752

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Cystine, an Essential Determinant of Protein Tertiary ...tools.thermofisher.com/content/sfs/posters/PN-71010-AOAC2014-Cystine... · Cystine, An Essential Determinant Of Protein Tertiary