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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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Waters, Micromass and Q-Tof micro are trademarks of Waters Corporation.All other trademarks are the property of their respective owners.©2004 Waters Corporation May 2004 720000871EN DE&LW-PDF
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