AUTOMATED CHIP-BASED NANOELECTROSPRAY

Advion BioSciences, Inc. (Ithaca, NY) has developeda method and demonstrated the capabilities of theN a n o M a t e™ system for quantitative determination ofnoncovalent interactions between proteins andligands. The NanoMate 100 is a chip-basedautomated nanoelectrospray ionization system formass spectro m e t ry, and is readily integrated with theWa t e r s® M i c ro m a s s® Q - Tof micro™.

Investigations of noncovalent pro t e i n - l i g e n dinteractions by nanoelectrospray ionization masss p e c t ro m e t ry (nanoESI/MS) are of great intere s tbecause of their relevance to molecular re c o g n i t i o nand to combinatory ligand library searching. Thisapplication note from Advion Biosciencesi n t roduces an experiment where automated nanoESI/MS analysis has been used to determ i n em i c romolar and submicromolar dissociationconstants as well as to measure the solution bidingconstants for the Ribonuclease A (RNase)complexes with cytidilic acid ligands.

S. Zhang, C.K. Van Pelt, D. B. WilsonAnal Chem 2003, 75,3010-3018

Q U A N T I TATIVE DETERMINATION OFN O N C O VALENT PROTEIN-LIGAND INTERACTIONS

A U T O M ATIC CHIP-BASED NANOELECTROSPRAY

Wa t e r s® M i c r o m a s s® Q - Tof micro™ Mass Spectrometer and the Advion NanoMate™ 1 0 0 .

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[RL]/[R] = 1/Kd * ( [Li ] - [RL] )

[R] * [L] [R] * ( [Li ] - [RL] )Kd = =

[RL] [RL ]

[R] * [L] [R] * ( [Li ] - [RL] )Kd = =

[RL] [RL ]

KdRL1= [R] * ( [R] +[RL 2] ) / [RL1]

KdRL2= [R] * ( [R] +[RL 1] ) / [RL2]

KdRL1= [R] * ( [R] +[RL 2] ) / [RL1]

KdRL2= [R] * ( [R] +[RL 1] ) / [RL2]

Titration Experiments

RNase protein was maintained at 10 µM and 4 µM, respectively, in10 mM ammonium acetate pH 6.8 for titration of 2’-CMP (1 to 20

µM) and CTP (1 to 8µM), respectively. The solutions were then

incubated at room temperature for 15 minutes prior to MS analysis.

Competitive Binding Experiments

Equimolar solutions of 2’-CMP and CTP (4 µM) were mixed with

4 µM of RNase in 10 mM ammonium acetate pH 6.8. Thesolutions were then incubated at room temperature for 15 minutes

prior to nanoelectrospray MS analysis.

NanoESI/MS Analytical Conditions

Sample Size: 3 µL

Flow Rate: 100 nL/minute

Spray Voltage: 1.5 kV

Pressure: 0.3 psi

Acquisition Time: 2 minutes

Instrumentation: NanoMateTM 100 with ESI ChipTM

Micromass Q-Tof microTM

Sample Cone Voltage: 30 V

Source temperature: 45 C

Quantitative Determination ofNoncovalent Protein-Ligand Interactions

NanoMateTM 100 Protein Application NMAP-004

Automated Chip-based Nanoelectrospray

Advion BioSciences, Inc. (Ithaca, NY)

has developed a method and

demonstrated the capabilities of the

NanoMateTM 100 system for

quantitative determination of

noncovalent interactions between

proteins and ligands. The NanoMate

100 is a chip-based automated

nanoelectrospray ionization system

for mass spectrometry.

Investigations of noncovalent protein-ligand interactions

by nanoelectrospray ionization mass spectrometry

(nanoESI/MS) are of great interest because of theirrelevance to molecular recognition and to combinatory

ligand library searching. The purpose of this

experiment is to demonstrate automated nanoESI/MSanalysis to determine micromolar and submicromolar

dissociation constants as well as to measure the solution

binding constants for the Ribonuclease A (RNase)complexes with cytidilic acid ligands.

Determination of Noncovalent Protein-

Ligand Interactions

RNase complexed with cytidine 2’-monophosphate and

cytidine triphosphate (see Figure 1), a well characterizedmodel system, was used to demonstrate the method.

O

OH

HH

HH

N

N

NH2

O

OH

OP

O

P

O

OOP

O

OH OHOH

O

O

HH

HH

N

N

NH2

O

OH

HO

PO

OH

OHHO

2’-CMP, MW 323.3CTP, MW 483.1

Calculations of dissociation constants:

Calculations of dissociation constants:

Figure 1 - Protein and Ligand Structures

RNase (1R0B.pdb)

MW 13682.0

y = 0.5633xR2 = 0.9836

0

1

2

3

4

5

0 2 4 6 8[Li] – [RL]

[RL]

/[R

]

Kd (µM)

Titration Experiment Ligand

Ave. of individual points

Plot

Competitive Binding Experiment

Cytidine 2’- monophosphate

(2’-CMP)

1.71 ± 0.33

2.00 ± 0.43

2.3 ± 0.4

Cytidine triphosphate (CTP)

0.80 ± 0.2

0.74 ± 0.3

0.75 ± 0.4

Table I - Summary of Binding Assay for RNase and Cytidine

Nucleotide Ligands Using Automated NanoESI/MS

13500 14000 14500mass0

100

%

13500 14000 14500mass0

100

%

13500 14000 14500mass0

100

%

100

0

100

0

100

0

13200 13800 14400Mass

13682.8

14005.8

14166.0

+ 323 Da

+ 483 Da

RNase

RNase + 2’CMP

RNase + CTP

1600 1800 2000 2200 2400m/z0

100

%

0

100

%

0

100

%

0

100

%8+

7+

6+

RNase

RNase + 2’-CMP

RNase + CTP

RNase + 2’-CMP + CTP

For more information, please contact:

Advion BioSciences, Inc.

15 Catherwood Rd.

Ithaca, NY 14850 USA

T: 607.266.0665

F: 607.266.0749

Advion BioSciences, Ltd.

Rowan House 26-28 Queens Rd.

Hethersett, Norwich, Norfolk NR9 3DB UK

T: +44 (0) 1603 813970

F: +44 (0) 1603 813971

info@advion.com

www.advion.com

Copyright © 2003 byAdvion BioSciences, Inc.

March 2003

Results

Each ligand was detected using the NanoMate 100 system(Figures 2and 3) and as a result titration and competitive

binding experiments were performed(Figure 4). The results

presented are in agreement with previously published results ofcircular dichroism (CD).1

Summary

The NanoMate 100 system can be used to determinemicromolar and submicromolar dissociation constants.

In addition, an automated nanoESI/MS method can be

used to measure solution binding constants for theRNase complexes with cytidilic acid ligands.

Advantages

• Allows extended acquisition time for better dataquality

• Increases sample throughput

• Improves spray stability and reproducibility

• Automates nanoelectrospray with one time sprayoptimization

• No carryover

1Jones, C.L.; Fish, F.; Muccio, D.D. Anal BioChem 2002, 302, 184-190.

Figure 4 - Titration Assay for RNase (10mM) with 2’-CMP

Figure 2 - NanoESI Mass Spectra

Figure 3 - Deconvoluted Mass Spectra of the

RNase-Ligand Complexes

Arrows indicate noncovalent protein-ligand complexes.

Rel

ativ

e Io

n A

bund

ance

%R

elat

ive

Ion

Abu

ndan

ce %

Application note based on

Zhang, S.; Van Pelt, C.K.; Wilson, D.B. Anal Chem 2003, 75, 3010-3018.

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