U.S. patent application number 09/276443 was filed with the patent office on 2001-06-14 for apparatus for screening compound libraries.
This patent application is currently assigned to OLE HINDSGAUL. Invention is credited to HINDSGAUL, OLE, SCHRIEMER, DAVID C..
Application Number | 20010003328 09/276443 |
Document ID | / |
Family ID | 26750531 |
Filed Date | 2001-06-14 |
United States Patent
Application |
20010003328 |
Kind Code |
A1 |
HINDSGAUL, OLE ; et
al. |
June 14, 2001 |
APPARATUS FOR SCREENING COMPOUND LIBRARIES
Abstract
Disclosed are apparatus for screening compound libraries using
frontal chromatography in combination with mass spectrometry to
identify and rank those members of the library that bind to a
target receptor. The apparatus of this invention also permit a
compound library to be rapidly screened to determine if any member
of the library has an affinity for the target receptor as measured
by a pre-selected indicator compound.
Inventors: |
HINDSGAUL, OLE; (ALBERTA,
CA) ; SCHRIEMER, DAVID C.; (ALBERTA, CA) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
OLE HINDSGAUL
|
Family ID: |
26750531 |
Appl. No.: |
09/276443 |
Filed: |
March 25, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09276443 |
Mar 25, 1999 |
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09069890 |
Apr 29, 1998 |
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6054047 |
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60079622 |
Mar 27, 1998 |
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Current U.S.
Class: |
210/198.2 |
Current CPC
Class: |
B01J 2219/00707
20130101; B01J 2219/00585 20130101; G01N 33/54306 20130101; G01N
33/538 20130101; B01J 2219/00704 20130101; G01N 30/466 20130101;
G01N 2030/628 20130101; B01J 2219/00731 20130101; G01N 30/7266
20130101; B01J 2219/00745 20130101; G01N 33/54366 20130101; G01N
33/68 20130101; B01J 2219/00747 20130101; G01N 33/6845 20130101;
C40B 30/08 20130101; G01N 33/6848 20130101; C40B 40/12 20130101;
G01N 33/6842 20130101; H01J 49/00 20130101; C40B 40/18 20130101;
B01J 2219/00738 20130101 |
Class at
Publication: |
210/198.2 |
International
Class: |
B01D 015/08 |
Claims
What is claimed is:
1. An apparatus for screening a compound library to determine the
relative or absolute affinity of a plurality of putative ligands to
a target receptor or a plurality of target receptors, which
apparatus comprises: (a) a column comprising a target receptor or a
plurality of target receptors, each target receptor optionally
attached to a solid phase support, and having a inflow end and an
outflow end, wherein said column is capable of having a compound
library comprising a plurality of putative ligands applied thereto
under frontal chromatography conditions to produce an effluent from
the outflow end of the column; (b) a first reservoir connected to
the inflow end of said column for applying the compound library to
the column; (c) a mass spectrometer connected to the outflow end of
said column for continuously or intermittently analyzing the
effluent from the column.
2. The apparatus of claim 1, wherein said apparatus further
comprises: (d) a second reservoir connected to the inflow end of
the column for applying either (i) a mixture comprising the
compound library, at least one void marker compound and an
indicator compound or a plurality of indicators compounds, (ii) at
least one void marker compound and an indicator compound or a
plurality of indicator compounds, or (iii) a buffer solution to the
column.
3. The apparatus of claim 1, wherein said apparatus further
comprises: (e) a third reservoir connected to the outflow end of
the column for supplying a supplemental diluent to the effluent
before analysis by the mass spectrometer.
4. The apparatus of claim 1, wherein the column has an internal
diameter ranging from about 10 .mu.m to about 4.6 mm.
5. The apparatus of claim 4, wherein the column has an internal
diameter of from about 100 .mu.m to about 250 .mu.m.
6. The apparatus of claim 1, wherein the column has a length of
from about 1 cm to about 30 cm.
7. The apparatus of claim 1, wherein the column has a length of
from about 2 cm to about 20 cm.
8. The apparatus of claim 1, wherein each target receptor is
independently selected from the group consisting of proteins,
glycoproteins, glycosaminoglycans, proteoglycans, integrins,
enzymes, lectins, selecting, cell-adhesion molecules, toxins,
bacterial pili, transport proteins, receptors involved in signal
transduction or hormone-binding, hormones, antibodies, major
histocompatability complexes, immunoglobulin superfamilies,
cadherins, DNA or DNA fragments, RNA and RNA fragments, whole
cells, cell fragments, tissues, bacteria, fungi, viruses,
parasites, preons, and synthetic analogs or derivatives
thereof.
9. The apparatus of claim 1, wherein the target receptor is bound
to a solid phase support.
10. The apparatus of claim 9, wherein the target receptor is
covalently bound to the solid phase support or bound via
biotin-avidin or biotin-streptavidin binding.
11. The apparatus of claim 9, wherein the solid phase support is
selected from the group consisting of polymeric beads, polymeric
gels, glass beads, silica chips, silica capillaries, agarose,
diatomaceous earths and pulp.
12. The apparatus of claim 1, wherein the column contains from
about 1 fmol to about 10 nmol of target receptor active sites.
13. The apparatus of claim 1, wherein the mass spectrometer is an
electrospray mass spectrometer.
14. An apparatus for screening a plurality of compound libraries to
determine the relative or absolute affinity of a plurality of
putative ligands in each library to a target receptor or a
plurality of target receptors, which apparatus comprises: (a) a
plurality of columns each column comprising a target receptor or a
plurality of target receptors, each target receptor optionally
attached to a solid phase support, and each column having a inflow
end and an outflow end, wherein each of said columns is capable of
independently having a compound library comprising a plurality of
putative ligands applied thereto under frontal chromatography
conditions to produce an effluent from the outflow end of the
column; (b) a plurality of first reservoirs each connected to the
inflow end of one of the columns for applying a compound library to
the columns; (c) a mass spectrometer connected to the outflow end
of each of said columns for intermittently analyzing the effluent
from each of the column.
15. The apparatus of claim 14, wherein said apparatus further
comprises: (d) a plurality of second reservoirs each connected to
the inflow end of one of the columns for applying either (i) a
mixture comprising the compound library, at least one void marker
compound and an indicator compound or a plurality of indicator
compounds, (ii) at least one void marker compound and an indicator
compound or a plurality of indicator compounds, or (iii) a buffer
solution to the column.
16. The apparatus of claim 14, wherein said apparatus further
comprises: (e) a third reservoir connected to the outflow end of
each of the columns for supplying a supplemental diluent to the
effluent from each column before analysis by the mass
spectrometer.
17. The apparatus of claim 14, wherein said apparatus comprises
from 2 to about 100 columns.
18. The apparatus of claim 17, wherein said apparatus comprises
from 3 to about 50 columns.
19. The apparatus of claim 18, wherein said apparatus comprises
from 5 to about 10 columns.
20. The apparatus of claim 14, wherein each column is
intermittently monitored for a period of about 0.5 seconds to about
10 seconds before switching to the next column.
21. The apparatus of claim 20, wherein each column is
intermittently monitored for about 1 second to about 5 seconds
before switching to the next column.
22. The apparatus of claim 14, wherein the column has an internal
diameter ranging from about 10 .mu.m to about 4.6 mm.
23. The apparatus of claim 22, wherein the column has an internal
diameter of from about 100 .mu.m to about 250 .mu.m.
24. The apparatus of claim 14, wherein the column has a length of
from about 1 cm to about 30 cm.
25. The apparatus of claim 14, wherein the column has a length of
from about 2 cm to about 20 cm.
26. The apparatus of claim 14, wherein each target receptor is
independently selected from the group consisting of proteins,
glycoproteins, glycosaminoglycans, proteoglycans, integrins,
enzymes, lectins, selectins, cell-adhesion molecules, toxins,
bacterial pili, transport proteins, receptors involved in signal
transduction or hormone-binding, hormones, antibodies, major
histocompatability complexes, immunoglobulin superfamilies,
cadherins, DNA or DNA fragments, RNA and RNA fragments, whole
cells, cell fragments, tissues, bacteria, fungi, viruses,
parasites, preons, and synthetic analogs or derivatives
thereof.
27. The apparatus of claim 14, wherein each target receptor is
bound to a solid phase support.
28. The apparatus of claim 27, wherein each target receptor is
covalently bound to the solid phase support or bound via
biotin-avidin or biotin-streptavidin binding.
29. The apparatus of claim 27, wherein the solid phase support is
selected from the group consisting of polymeric beads, polymeric
gels, glass beads, silica chips, silica capillaries, agarose,
diatomaceous earths and pulp.
30. The apparatus of claim 14, wherein the column contains from
about 1 fmol to about 10 nmol of target receptor active sites.
31. The apparatus of claim 14, wherein the mass spectrometer is an
electrospray mass spectrometer.
32. An apparatus for screening a target receptor or a plurality of
target receptors to determine the relative affinity of the receptor
or receptors to an immobilized ligand or ligands relative to an
indicator compound or a plurality of indicator compounds, which
apparatus comprises: (a) a column comprising a ligand or a
plurality of ligands wherein each ligand is bound to a solid phase
support, said column having a inflow end and an outflow end and
further wherein said column is capable of having a target receptor
or a plurality of target receptors applied thereto under frontal
chromatography conditions to produce an effluent from the outflow
end of the column; (b) a first reservoir connected to the inflow
end of said column for applying the target receptor or receptors to
the column; (c) a second reservoir connected to the inflow end of
the column for applying either (i) a mixture comprising the target
receptor or receptors, at least one void marker compound and an
indicator compound or a plurality of indicators compounds, (ii) at
least one void marker compound and an indicator compound or a
plurality of indicator compounds, or (iii) a buffer solution to the
column; (d) a mass spectrometer connected to the outflow end of
said column for continuously or intermittently analyzing the
effluent from the column.
33. The apparatus of claim 32, wherein said apparatus further
comprises: (e) a third reservoir connected to the outflow end of
the column for supplying a supplemental diluent to the effluent
before analysis by the mass spectrometer.
34. The apparatus of claim 32, wherein the column has an internal
diameter ranging from about 10 .mu.m to about 4.6 mm.
35. The apparatus of claim 34, wherein the column has an internal
diameter of from about 100 .mu.m to about 250 .mu.m.
36. The apparatus of claim 32, wherein the column has a length of
from about 1 cm to about 30 cm.
37. The apparatus of claim 32, wherein the column has a length of
from about 2 cm to about 20 cm.
38. The apparatus of claim 32, wherein each ligand is selected from
the group consisting of carbohydrates, monosaccharides,
oligosaccharides, polysaccharides, amino acids, peptides,
oligopeptides, polypeptides, proteins, nucleosides, nucleotides,
oligonucleotides, polynucleotides, lipids, retinoids, steroids,
glycopeptides, glycoproteins, glycolipids, proteoglycans, and
synthetic analogs or derivatives thereof.
39. The apparatus of claim 32, wherein each ligand is selected from
the group consisting of synthetic small molecule organic
compounds.
40. An apparatus for screening a plurality of target receptors to
determine the relative affinity of the receptors to an immobilized
ligand or ligands relative to an indicator compound or a plurality
of indicator compounds, which apparatus comprises: (a) a plurality
of columns each column comprising a ligand or a plurality of
ligands wherein each ligand is bound to a solid phase support, and
each column having a inflow end and an outflow end, wherein each of
said columns is capable of independently having a target receptor
or a plurality of target receptors applied thereto under frontal
chromatography conditions to produce an effluent from the outflow
end of the column; (b) a plurality of first reservoirs each
connected to the inflow end of one of the columns for applying a
target receptor or a plurality of target receptors to the columns;
(c) a plurality of second reservoirs each connected to the inflow
end of one of the columns for applying either (i) a mixture
comprising the target receptor or plurality of target receptors, at
least one void marker compound and an indicator compound or a
plurality of indicator compounds, (ii) at least one void marker
compound and an indicator compound or a plurality of indicator
compounds, or (iii) a buffer solution to the column; (d) a mass
spectrometer connected to the outflow end of each of said columns
for intermittently analyzing the effluent from each of the
column.
41. The apparatus of claim 40, wherein said apparatus further
comprises: (e) a third reservoir connected to the outflow end of
each of the columns for supplying a supplemental diluent to the
effluent from each column before analysis by the mass
spectrometer.
42. The apparatus of claim 40, wherein said apparatus comprises
from 2 to about 100 columns.
43. The apparatus of claim 42, wherein said apparatus comprises
from 3 to about 50 columns.
44. The apparatus of claim 43, wherein said apparatus comprises
from 5 to about 10 columns.
45. The apparatus of claim 40, wherein each column is
intermittently monitored for a period of about 0.5 seconds to about
10 seconds before switching to the next column.
46. The apparatus of claim 45, wherein each column is
intermittently monitored for about 1 second to about 5 seconds
before switching to the next column.
47. The apparatus of claim 40, wherein the column has an internal
diameter ranging from about 10 .mu.m to about 4.6 mm.
48. The apparatus of claim 47, wherein the column has an internal
diameter of from about 100 .mu.m to about 250 .mu.m.
49. The apparatus of claim 40, wherein the column has a length of
from about 1 cm to about 30 cm.
50. The apparatus of claim 40, wherein the column has a length of
from about 2 cm to about 20 cm.
51. The apparatus of claim 40, wherein each ligand is selected from
the group consisting of carbohydrates, monosaccharides,
oligosaccharides, polysaccharides, amino acids, peptides,
oligopeptides, polypeptides, proteins, nucleosides, nucleotides,
oligonucleotides, polynucleotides, lipids, retinoids, steroids,
glycopeptides, glycoproteins, glycolipids, proteoglycans, and
synthetic analogs or derivatives thereof.
52. The apparatus of claim 40, wherein each ligand is selected from
the group consisting of synthetic small molecule organic compounds.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
09/069,890, filed Apr. 29, 1998, which application claims the
benefit of U.S. Provisional Application No. 60/079,622, filed Mar.
27, 1998. Each of these applications are incorporated herein by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to apparatus for screening compound
libraries, such as compound libraries generated using combinatorial
chemistry techniques. The apparatus of this invention employ
frontal chromatography in combination with mass spectrometry to
screen a library of compounds to identify and rank those members of
the library that bind to a target receptor. The apparatus of this
invention also permit a compound library to be rapidly screened to
determine if one or more members of the library have an affinity
for a target receptor as measured by a pre-selected indicator
compound.
[0004] 2. References
[0005] The following publications, patents and patent applications
are cited in this application as superscript numbers:
[0006] .sup.1 K. S. Lam, Anti-Cancer Drug Des. 1997, 12,
145-167.
[0007] .sup.2 P. M. Sweetnam et al., In Burger's Medicinal
Chemistry and Drug Discovery; M. E. Wolff, Ed.; John Wiley &
Sons: New York, 1995; pp 697-731.
[0008] .sup.3 R. H. Griffey et al., In Proceedings of the 45.sup.th
ASMS Conference on Mass Spectrometry and Allied Topics, Palm
Springs, Calif., Jun. 1-5, 1997; p. 400.
[0009] .sup.4 L. Fang et al., In Proceedings of the 45.sup.th ASMS
Conference on Mass Spectrometry and Allied Topics, Palm Springs,
Calif., Jun. 1-5, 1997; p. 401.
[0010] .sup.5 Y.-H. Chu et al., J. Am. Chem. Soc. 1996, 118,
7827-7835.
[0011] .sup.6 Y.-Z. Zhao et al., J. Med. Chem. 1997, 40,
4006-4012.
[0012] .sup.7 Y. F. Hsieh et al., J. Mol. Div. 1996, 2,
189-196.
[0013] .sup.8 R. W. Nelson et al., Anal. Chem. 1995, 67,
1153-1158.
[0014] .sup.9 D. C. Schriemer and L. Li, Anal. Chem. 1996, 68,
3382-3387.
[0015] .sup.10 PCT/US97/07964 (International Publication No. WO
97/43641), published Nov. 20, 1997, entitled "Molecular Diversity
Screening Device and Method."
[0016] .sup.11 R. Wieboldt et al., Anal. Chem. 1997, 69,
1683-1691.
[0017] .sup.12 R. B. van Breemen et al., Anal. Chem. 1997, 69,
2159-2164.
[0018] .sup.13 M. L. Nedved et al., Anal. Chem. 1996, 68,
4228-4236.
[0019] .sup.14 PCT/US95/03355 (International Publication No. WO
95/25737), published Sep. 28, 1995, entitled "Method for
Identifying Members of Combinatorial Libraries."
[0020] .sup.15 PCT/EP97/02215 (International Publication No. WO
97/43301), published Nov. 20, 1997, entitled "Identification of
Members of Combinatorial Libraries By Mass Spectrometry."
[0021] All of the above publications, patents and patent
applications are herein incorporated by reference in their entirety
to the same extent as if each individual publication, patent or
patent application was specifically and individually indicated to
be incorporated by reference in its entirety.
[0022] State of the Art
[0023] In recent years, a large number of combinatorial chemistry
techniques have been developed which permit vast libraries of
diverse chemical compounds to be rapidly synthesized..sup.1 In
combinatorial chemistry, a series of chemical reactions is
typically conducted employing a plurality of reagents at each step
to generate a library of compounds. Such techniques have the
potential to greatly accelerate the discovery of new compounds
having biologically useful properties by providing large
collections of diverse chemical compounds for biological
screening.
[0024] This ability to rapidly generate large collections of
compounds using combinatorial chemistry techniques has created a
need for new methods of screening compound libraries. The
traditional approach of screening each compound individually in an
assay to identify those compounds having the desired biological
activity is no longer practical due to time and resource
constraints. Thus, a need exists for new methods and apparatus
which permit the rapid screening of compound libraries.
[0025] In this regard, various methods for screening compound
libraries have been reported. Typically, these screening methods
involve the use of target receptors which have been labeled with
fluorescent or other reporter groups..sup.2 In these methods, the
compound library, typically bound to a resin bead, is exposed to
the labeled target receptor and those members binding to the
labeled target receptor are identified and physically separated.
The particular ligand binding to the target receptor is then
identified. In many of these techniques, elaborate procedures are
required to keep track of individual members of the library. For
example, coded tags are often added during the synthesis of the
combinatorial library to allow the structure of the individual
members to be subsequently determined. Alternatively, combinatorial
libraries can be prepared in an array and the individual members of
the library subsequently identified by their location in the array.
While such methods can be effective, the need to keep track of
individual members of the library during their synthesis and
screening is quite cumbersome and often limits the type of
synthetic procedures that can be employed. Additionally, many of
these techniques require that the synthetic procedures be conducted
on a solid phase, thus further limiting the synthetic procedures
and reagents that can be used.
[0026] As an alternative, mass spectrometry is emerging as an
important tool for the interrogation of combinatorial libraries. To
date, mass spectrometry has been used to assess library
quality.sup.3,4 and, when coupled with molecular recognition
technologies, has allowed for some success in the isolation and
characterization of active library compounds..sup.5-15 Typically,
when screening compound libraries for biologically active members,
mass spectrometry is used in combination with a "capture and
release" methodology. In this methodology, compound mixtures are
presented to the target receptor, which is often immobilized on a
solid support, and the resulting ligand-receptor complexes are
separated from the unbound members of the library. After
separation, the ligand-receptor complexes are typically denatured,
for example, with a solvent and the solvent mixture containing the
previously bound ligands is presented to the mass spectrometer to
permit identification of the high affinity ligands.
[0027] For example, ultrafiltration has been used in combination
with electrospray mass spectrometry to screen combinatorial
libraries..sup.10-12 In this method, ligands present in a compound
library are allowed to bind to a receptor and the resulting
ligand-receptor complexes are purified by ultrafiltration. The
ligand-receptor complexes are then dissociated using a solvent,
such as methanol, and the previously bound ligands are detected by
an electrospray mass spectrometer.
[0028] Affinity capillary electrophoresis (ACE) has also been
coupled with mass spectrometry to screen combinatorial
libraries..sup.5 In this procedure, ACE is used to separate
ligand-receptor complexes from unbound ligands and mass
spectrometry is used to identify the high affinity ligands.
[0029] Similarly, compound libraries have been screened using
affinity chromatography in combination with mass spectrometry. For
example, WO 97/43301 describes a method for characterizing the
members of a combinatorial library, which method utilizes affinity
selection in combination with mass spectrometry. Specifically, the
members of the library are brought into contact with a domain of
interest to allow for binding, i.e., the formation of a complex.
After binding, the complex is separated from the unbound members of
the library, typically by washing the unbound members from the
column containing the complexes. The complexes are then treated to
elute the bound library components and the eluted components are
analyzed by mass spectrometry. The elution methods described
include the use of displacers, chaotrope agents, pH elution, salt
gradients, temperature gradients, organic solvents, selective
denaturants and detergents. Using such methods, the weakly bound
members of the library are purportedly eluted first and analyzed by
mass spectrometry, followed by the elution of the more strongly
bound members.
[0030] There are several disadvantages associated with the "capture
and release" methods for screening compound libraries that have
been previously reported. First, the procedure used to "release"
the bound ligands from the ligand-receptor complexes may alter the
binding profile for the various bound ligands, resulting in a false
indication of binding strength. For example, using a pH gradient to
release the bound members of the library may change the electronic
character of the binding site on the receptor causing ligands which
are strongly bound under physiological conditions to be prematurely
released. Thus, the characterization of binding strength for
various ligands based on their relative time of release may be
misleading if the release conditions are different from the binding
conditions. Accordingly, these methods may not accurately identify
the most active members of a compound library. Additionally,
certain conditions used for compound release, such as pH gradients,
may irreversibly denature the receptor thus preventing its
subsequent use for screening compound libraries.
[0031] Additionally, when "capture and release" methods are
employed, each bound ligand is typically released over a relatively
short period of time resulting, for example, in an elution peak or
"spike" for each ligand. Accordingly, the effluent produced using
such methods is typically monitored continually, for example, by
mass spectrometry so that any particular elution peak is not
missed. Thus, the number of analyses that can be conducted using
any particular mass spectrometer is limited. Accordingly, it would
be desirable to develop methods and apparatus for screening
compound libraries that do not rely upon "capture and release"
methodologies.
SUMMARY OF THE INVENTION
[0032] This invention is directed to apparatus for screening
compound libraries. The compound libraries may be generated or
obtained by any means including, by way of example, combinatorial
chemistry techniques or from fermentation broths, plant extracts,
cellular extracts and the like. The apparatus of this invention
employ frontal chromatography (FC) in combination with mass
spectrometry (MS) to screen the library of compounds to identify
and rank those members of the library that bind to a target
receptor.
[0033] In frontal chromatography, a target receptor is typically
immobilized on a suitable solid support material and packed in a
column. A mixture containing putative ligands is then continuously
infused through the column. Ligands having an affinity for the
target receptor bind to the column, but eventually the capacity of
the column for each ligand is exceeded and the ligands elute or
"break through" at their infusion concentration. Once a ligand
begins eluting from the column, it is continually present in the
effluent. Compounds having little or no affinity for the target
receptor break through earlier in the effluent compared to ligands
having a higher affinity for the receptor.
[0034] In the present invention, mass spectrometry (MS) is employed
to continuously or intermittently monitor the FC effluent. Using
MS, the identity and break through time for each ligand on the
column can be determined. Thus, FC-MS allows the relative affinity
of each member of the library for the target receptor to be
determined relative to other members of the library under
ligand-receptor binding conditions. Using the present apparatus, an
accurate ranking of the relative affinity of each member of the
compound library for the target receptor can be ascertained.
[0035] Accordingly, in one of its apparatus aspects, the present
invention is directed to an apparatus for screening a compound
library to determine the relative or absolute affinity of a
plurality of putative ligands to a target receptor or a plurality
of target receptors, which apparatus comprises:
[0036] (a) a column comprising a target receptor or a plurality of
target receptors, each target receptor optionally attached to a
solid phase support, and having a inflow end and an outflow end,
wherein said column is capable of having a compound library
comprising a plurality of putative ligands applied thereto under
frontal chromatography conditions to produce an effluent from the
outflow end of the column;
[0037] (b) a first reservoir connected to the inflow end of said
column for applying the compound library to the column;
[0038] (c) a mass spectrometer connected to the outflow end of said
column for continuously or intermittently analyzing the effluent
from the column.
[0039] In a preferred embodiment, the above described apparatus
further comprises:
[0040] (d) a second reservoir connected to the inflow end of the
column for applying either (i) a mixture comprising the compound
library, at least one void marker compound and an indicator
compound or a plurality of indicators compounds, (ii) at least one
void marker compound and an indicator compound or a plurality of
indicator compounds, or (iii) a buffer solution to the column.
[0041] In another preferred embodiment, the above described
apparatus further comprises:
[0042] (e) a third reservoir connected to the outflow end of the
column for supplying a supplemental diluent to the effluent before
analysis by the mass spectrometer.
[0043] Preferably, the column employed in this invention will have
an internal diameter (i.d.) ranging from about 10 .mu.m to about
4.6 mm. More preferably, the internal diameter of the column will
be in the range of from about 100 .mu.m to about 250 .mu.m.
[0044] Preferably, the column will range in length from about 1 cm
to about 30 cm, more preferably from about 2 cm to about 20 cm.
[0045] Preferably, each target receptor is independently selected
from the group consisting of proteins, including recombinant
proteins, glycoproteins, glycosaminoglycans, proteoglycans,
integrins, enzymes, lectins, selecting, cell-adhesion molecules,
toxins, bacterial pili, transport proteins, receptors involved in
signal transduction or hormone-binding, hormones, antibodies, major
histocompatability complexes, immunoglobulin superfamilies,
cadherins, DNA or DNA fragments, RNA and RNA fragments, whole
cells, cell fragments, tissues, bacteria, fungi, viruses,
parasites, preons, and synthetic analogs or derivatives
thereof.
[0046] Additionally, each target receptor is preferably bound to a
solid phase support. More preferably, each target receptor is
covalently bound to the solid phase support or bound via
biotin-avidin or biotin-streptavidin binding.
[0047] Preferably, the solid phase support used in this invention
is selected from the group consisting of polymeric (resin) beads,
polymeric gels, glass beads, silica chips, silica capillaries,
agarose, diatomaceous earths and pulp.
[0048] The column employed in this invention preferably contains
from about 1 fmol to about 10 nmol of target receptor active sites;
preferably, from about 1 pmol to about 10 nmol of target receptor
active sites.
[0049] Preferably, the mass spectrometer employed in this invention
is an electrospray mass spectrometer.
[0050] Additionally, since FC-MS does not require constant effluent
monitoring, a plurality of FC-MS analyses can be conducted
simultaneously using a single mass spectrometer to intermittently
monitor each column. Furthermore, under FC conditions, since
ligands are always present in the effluent once they break through
the column, the intermittent monitoring of each column does not
necessarily require monitoring the actual break through time for
each ligand. Therefore, a plurality of FC-MS analyses can be
conducted simultaneously using a single mass spectrometer to
intermittently monitor each column.
[0051] Accordingly, in another of its apparatus aspects, this
invention provides an apparatus for screening a plurality of
compound libraries to determine the relative or absolute affinity
of a plurality of putative ligands in each library to a target
receptor or a plurality of target receptors, which apparatus
comprises:
[0052] (a) a plurality of columns each column comprising a target
receptor or a plurality of target receptors, each target receptor
optionally attached to a solid phase support, and each column
having a inflow end and an outflow end, wherein each of said
columns is capable of independently having a compound library
comprising a plurality of putative ligands applied thereto under
frontal chromatography conditions to produce an effluent from the
outflow end of the column;
[0053] (b) a plurality of first reservoirs each connected to the
inflow end of one of the columns for applying a compound library to
the columns;
[0054] (c) a mass spectrometer connected to the outflow end of each
of said columns for intermittently analyzing the effluent from each
of the column.
[0055] In a preferred embodiment, the above described apparatus
further comprises:
[0056] (d) a plurality of second reservoirs each connected to the
inflow end of one of the columns for applying either (i) a mixture
comprising the compound library, at least one void marker compound
and an indicator compound or a plurality of indicator compounds,
(ii) at least one void marker compound and an indicator compound or
a plurality of indicator compounds, or (iii) a buffer solution to
the column.
[0057] In another preferred embodiment, the above described
apparatus further comprises:
[0058] (e) a third reservoir connected to the outflow end of each
of the columns for supplying a supplemental diluent to the effluent
from each column before analysis by the mass spectrometer.
[0059] Preferably, the above described apparatus comprises from 2
to about 100 columns, more preferably from 3 to about 50 columns;
and still more preferably from 5 to about 10 columns.
[0060] Preferably, each column is intermittently monitored for a
period of about 0.5 seconds to about 10 seconds, preferably for
about 1 second to about 5 seconds, before switching to the next
column.
[0061] The apparatus of this invention can also be employed to
screen a target receptor or a plurality of target receptors for
affinity to an immobilized ligand or plurality of ligands. This
embodiment is particularly useful for target validation studies on
ligands having biological effects. Accordingly, in another of its
apparatus aspects, this invention provides an apparatus for
screening a target receptor or a plurality of target receptors to
determine the relative affinity of the receptor or receptors to an
immobilized ligand or ligands relative to an indicator compound or
a plurality of indicator compounds, which apparatus comprises:
[0062] (a) a column comprising a ligand or a plurality of ligands
wherein each ligand is bound to a solid phase support, said column
having a inflow end and an outflow end and further wherein said
column is capable of having a target receptor or a plurality of
target receptors applied thereto under frontal chromatography
conditions to produce an effluent from the outflow end of the
column;
[0063] (b) a first reservoir connected to the inflow end of said
column for applying the target receptor or receptors to the
column;
[0064] (c) a second reservoir connected to the inflow end of the
column for applying either (i) a mixture comprising the target
receptor or receptors, at least one void marker compound and an
indicator compound or a plurality of indicators compounds, (ii) at
least one void marker compound and an indicator compound or a
plurality of indicator compounds, or (iii) a buffer solution to the
column.
[0065] (d) a mass spectrometer connected to the outflow end of said
column for continuously or intermittently analyzing the effluent
from the column.
[0066] In a preferred embodiment, the above apparatus further
comprises:
[0067] (e) a third reservoir connected to the outflow end of the
column for supplying a supplemental diluent to the effluent before
analysis by the mass spectrometer.
[0068] In a preferred embodiment, each ligand employed in the above
apparatus is selected from the group consisting of carbohydrates,
monosaccharides, oligosaccharides, polysaccharides, amino acids,
peptides, oligopeptides, polypeptides, proteins, nucleosides,
nucleotides, oligonucleotides, polynucleotides, lipids, retinoids,
steroids, glycopeptides, glycoproteins, glycolipids, proteoglycans,
and synthetic analogs or derivatives thereof.
[0069] In another preferred embodiment, each ligand is selected
from the group consisting of synthetic small molecule organic
compounds.
[0070] A plurality of such FC-MS analyses can also be conducted
simultaneously using a single mass spectrometer to intermittently
monitor each column. Accordingly, in yet another of its apparatus
aspects, the present invention provides an apparatus for screening
a plurality of target receptors to determine the relative affinity
of the receptors to an immobilized ligand or ligands relative to an
indicator compound or a plurality of indicator compounds, which
apparatus comprises:
[0071] (a) a plurality of columns each column comprising a ligand
or a plurality of ligands wherein each ligand is bound to a solid
phase support, and each column having a inflow end and an outflow
end, wherein each of said columns is capable of independently
having a target receptor or a plurality of target receptors applied
thereto under frontal chromatography conditions to produce an
effluent from the outflow end of the column;
[0072] (b) a plurality of first reservoirs each connected to the
inflow end of one of the columns for applying a target receptor or
a plurality of target receptors to the columns;
[0073] (c) a plurality of second reservoirs each connected to the
inflow end of one of the columns for applying either (i) a mixture
comprising the target receptor or plurality of target receptors, at
least one void marker compound and an indicator compound or a
plurality of indicator compounds, (ii) at least one void marker
compound and an indicator compound or a plurality of indicator
compounds, or (iii) a buffer solution to the column.
[0074] (d) a mass spectrometer connected to the outflow end of each
of said columns for intermittently analyzing the effluent from each
of the column.
[0075] In a preferred embodiment, the above apparatus further
comprises:
[0076] (e) a third reservoir connected to the outflow end of each
of the columns for supplying a supplemental diluent to the effluent
from each column before analysis by the mass spectrometer.
[0077] Preferably, the above described apparatus comprises from 2
to about 100 columns, more preferably from 3 to about 50 columns;
and still more preferably from 5 to about 10 columns.
[0078] Preferably, each column is intermittently monitored for a
period of about 0.5 seconds to about 10 seconds, preferably for
about 1 second to about 5 seconds, before switching to the next
column.
BRIEF DESCRIPTION OF THE DRAWINGS
[0079] FIG. 1 illustrates a representative apparatus for screening
compound libraries using frontal chromatography in combination with
a mass spectrometer.
[0080] FIG. 2 illustrates a representative apparatus for screening
compound libraries using a plurality of frontal chromatography
columns in combination with a mass spectrometer.
[0081] FIG. 3 illustrates another representative apparatus for
screening compound libraries using a plurality of frontal
chromatography columns in combination with a mass spectrometer.
[0082] FIG. 4 illustrates a representative apparatus for
sequentially screening compound libraries with an indicator
compound using a plurality of frontal chromatography columns in
combination with a mass spectrometer.
[0083] FIG. 5A shows a total ion chromatogram (TIC) from a FC-MS
run using six representative oligosaccharides having varying
affinity for a carbohydrate-binding antibody that recognizes the
3,6-dideoxy-D-galactose (abequose) epitope in Salmonella paratyphi
B O-antigens.
[0084] FIG. 5B shows selected ion chromatograms for the six
oligosaccharides reconstructed from the TIC shown in FIG. 5A.
[0085] FIGS. 5C, 5D and 5E show mass spectra generated from
time-slices of the TIC shown in FIG. 5A.
[0086] FIG. 6 shows a plot of ([A].sub.0(V-V.sub.0)).sup.-1 versus
[A].sub.0.sup.-1 for
.alpha.Gal(1.fwdarw.2)[.alpha.Abe(1.fwdarw.3)].alpha-
.Man-OCH.sub.3.
[0087] FIG. 7A shows a selected ion chromatogram from a FC-MS run
using an indicator compound in the absence of a compound
library.
[0088] FIG. 7B shows a selected ion chromatogram from a FC-MS run
using an indicator compound in the presence of a compound
library.
[0089] FIG. 8 shows a selected ion chromatogram from a FC-MS run
using four representative oligosaccharides having varying affinity
for cholera toxin B subunit.
[0090] FIG. 9 shows a selected ion chromatogram from a FC-MS run
using a synthetically prepared GM.sub.1 analog.
[0091] FIG. 10 illustrates the "roll-up" effect in a selected ion
chromatogram from a FC-MS run using an indicator compound in the
presence of a compound library.
[0092] FIG. 11 is a graph of the reduction of column activity as a
function of time for two different compound libraries, the first
library containing many weak binders and the second library
containing strong binders.
[0093] FIG. 12A shows schematically the synthesis of a compound
library containing 100 tripeptides. FIG. 12B shows an electrospray
mass spectrum of the compound library.
[0094] FIG. 13 shows a chromatogram of three infusion/wash cycles
of a compound library containing 100 tripeptides, an indicator
compound (dashed line) and a void marker compound (solid line).
[0095] FIG. 14A illustrates the V-V.sub.0 value for an indicator
compound (dashed line) relative to a void marker compound (solid
line) immediately before equilibration of a column with a compound
library containing 100 tripeptides. FIG. 14B illustrates the
V-V.sub.0 value for the indicator compound immediately after
equilibration of the column with the compound library.
[0096] FIG. 15A shows a total ion chromatogram for a compound
library containing 100 tripeptides. FIG. 15B shows a selected ion
chromatogram for m/z 419.2.
[0097] FIG. 16A shows a selected ion chromatogram for fPR. FIG. 16B
shows a selected ion chromatogram of fPR and fPR-chloromethyl
ketone.
DETAILED DESCRIPTION OF THE INVENTION
[0098] The present invention provides apparatus for screening
compound libraries using frontal chromatography in combination with
mass spectrometry. When describing the apparatus of this invention,
the following terms have the following meanings, unless otherwise
indicated. All terms not defined herein have their conventional
art-recognized meaning.
[0099] Definitions
[0100] The term "buffer" refers to a solution that stabilizes the
binding activity of the target receptor. Typical buffers include,
by way of illustration, pH buffers and buffers containing specific
molecules, either organic or inorganic, required to stabilize the
binding activity of a specific target receptor.
[0101] The term "break through time" refers to the period of time
between elution of the void volume and the front corresponding to
the elution of a particular compound during frontal chromatography.
The term "break through curve" refers to the signal intensity as a
function of time resulting from the infusion of compound(s) through
a column under frontal chromatography conditions. Typically, the
break through curve is comprised of a front (fast-rising section)
and a plateau (horizontal section).
[0102] The term "compound library" refers to a mixture or
collection of one or more putative ligands generated or obtained in
any manner. Preferably, the library contains more than one putative
ligand or member.
[0103] The term "electrospray" refers to the generation of
gas-phase ions from a flowing solution. Electrospray is typically
performed at atmospheric pressure in an electric field with or
without assisted nebulization and solvent evaporation.
[0104] The term "effluent" refers to the solvent or solution
emerging or exiting from the frontal chromatography column.
[0105] The term "frontal chromatography conditions" refers to
chromatography conditions in which a solution of compounds, such as
putative ligands and/or indicator compounds, is applied or infused
through a column to generate a break through curve. Typically,
under frontal chromatography conditions, putative ligands are
infused continuously at a constant concentration through a column
containing a target receptor such that the target receptor is
continuously contacted with the putative ligands during the
chromatography.
[0106] The term "indicator compound" or "indicator" refers to a
compound having a known affinity or specificity for the target
receptor and a measurable break through time under frontal
chromatography conditions. When screening target receptors for
affinity to a particular ligand(s), the indicator may also be a
compound, such as a protein, or other biological entity, such as a
cell or cell fragment, having a known affinity or specificity for
the ligand(s). The break through time for the indicator is
typically referenced to the void volume of the system.
[0107] The term "ligand" refers to a molecule or group of molecules
that bind to one or more specific sites of a receptor.
Representative ligands include, by way of illustration,
carbohydrates, monosaccharides, oligosaccharides, polysaccharides,
amino acids, peptides, oligopeptides, polypeptides, proteins,
nucleosides, nucleotides, oligonucleotides, polynucleotides,
including DNA and DNA fragments, RNA and RNA fragments and the
like, lipids, retinoids, steroids, glycopeptides, glycoproteins,
glycolipids, proteoglycans and the like, and synthetic analogues or
derivatives thereof, including peptidomimetics, natural products or
naturally-occurring small molecule organic compounds (i.e.,
compounds produced by and/or isolated from natural sources, such as
soil, water, cells, plants, fungi, animals and the like), synthetic
small molecule organic compounds, inorganic ions, organometallic
compounds and the like, and mixtures thereof. The term "putative
ligand" refers to a ligand whose affinity or specificity for a
target receptor, if any, has not been determined.
[0108] The term "microcolumn" refers to a column having an internal
diameter less than or equal to about 1 mm.
[0109] The term "natural products" refers to compounds isolated
from natural sources, such as cells, plants, fungi, animals and the
like. The term "naturally-occurring small molecule organic
compounds" refers to natural products that are organic compounds
generally having a molecular weight less than about 1000,
preferably less than about 500.
[0110] The term "selected ion chromatogram" refers to a plot of ion
abundance vs. time constructed from the intensity of a single ion.
A selected ion chromatogram can be prepared from a scan or selected
ion monitoring mode.
[0111] The term "selected ion monitoring" refers to the detection
of a few pre-selected ions using a mass spectrometer (e.g.
quadrupoles).
[0112] The term "signal intensity" refers to the measured response
of an instrument to a stimulus, for example, the current output of
a high energy dynode detector resulting from the impact of an
ion.
[0113] The term "solid support" or "solid phase support" refers to
an inert material or molecule to which a target receptor may be
bound or coupled, either directly or through a linking arm.
[0114] The term "synthetic small molecule organic compounds" refers
to organic compounds generally having a molecular weight less than
about 1000, preferably less than about 500, which are prepared by
synthetic organic techniques, such as by combinatorial chemistry
techniques.
[0115] The term "supplemental diluent" or "make-up flow" refers to
a solution or solvent which is combined with the effluent from a
column before the effluent passes through the mass analyzer of a
mass spectrometer.
[0116] The term "target receptor" or "receptor" refers to a
molecule or a group of molecules capable of binding a ligand at a
specific site. Representative examples of target receptors include,
by way of example, proteins, including recombinant proteins,
glycoproteins, glycosaminoglycans, proteoglycans, integrins,
enzymes, lectins, selecting, cell-adhesion molecules, toxins,
bacterial pili, transport proteins, receptors involved in signal
transduction or hormone-binding, hormones, antibodies, major
histocompatability complexes, immunoglobulin superfamilies,
cadherins, DNA or DNA fragments, RNA and RNA fragments, whole
cells, cell fragments, tissues, bacteria, fungi, viruses,
parasites, preons, and synthetic analogs or derivatives
thereof.
[0117] The term "target receptor active site" refers to the binding
site of interest on a particular target receptor.
[0118] The term "total ion chromatogram" refers to a plot of ion
abundance vs. time constructed from a summation of all ion
intensities in a scan. In a total ion chromatogram, the number of
scans are linearly related to time.
[0119] The term "void marker compound" or "void marker" refers to a
compound that elutes from column at the void volume. The void
marker compound is used to identify the void volume of a column
used under frontal chromatography conditions.
[0120] The term "void volume" or "V.sub.0" refers to the volume of
solution which passes through a frontal chromatography column from
the point of infusion to the point of detection of a compound, i.e.
a putative ligand, in the absence (or suppression) of a binding
event. Since the flow rate is typically constant, void volume is
generally specified in terms of a retention time for the compound.
Putative ligands having no affinity for the target receptor
typically elute from column at the void volume.
[0121] The compound libraries employed in this invention may be
prepared or obtained by any means including, but not limited to,
combinatorial chemistry techniques, fermentation methods, plant and
cellular extraction procedures and the like. Methods for making
combinatorial libraries are well-known in the art. See, for
example, E. R. Felder, Chimia 1994, 48, 512-541; Gallop et al., J.
Med. Chem. 1994, 37, 1233-1251; R. A. Houghten, Trends Genet. 1993,
9, 235-239; Houghten et al., Nature 1991, 354, 84-86; Lam et al.,
Nature 1991, 354, 82-84; Carell et al., Chem. Biol. 1995, 3,
171-183; Madden et al., Perspectives in Drug Discovery and Design
2, 269-282; Cwirla et al., Biochemistry 1990, 87, 6378-6382;
Brenner et al., Proc. Natl. Acad. Sci. USA 1992, 89, 5381-5383;
Gordon et al., J. Med. Chem. 1994, 37, 1385-1401; Lebl et al.,
Biopolymers 1995, 37 177-198; and references cited therein. Each of
these references is incorporated herein by reference in its
entirety.
[0122] Any type of molecule that is capable of binding to a target
receptor may be present in the compound library. For example,
compound libraries screened using this invention may contain
naturally-occurring molecules, such as carbohydrates,
monosaccharides, oligosaccharides, polysaccharides, amino acids,
peptides, oligopeptides, polypeptides, proteins, nucleosides,
nucleotides, oligonucleotides, polynucleotides, including DNA and
DNA fragments, RNA and RNA fragments and the like, lipids,
retinoids, steroids, glycopeptides, glycoproteins, glycolipids,
proteoglycans and the like; or analogs or derivatives of
naturally-occurring molecules, such peptidomimetics and the like;
and non-naturally occurring molecules, such as "small molecule"
organic compounds generated, for example, using combinatorial
chemistry techniques; organometallic compounds, inorganic ions, and
mixtures thereof. The term "small molecule organic compound" refers
to organic compounds generally having a molecular weight less than
about 1000, preferably less than about 500.
[0123] A particular advantage of FC-MS is that compound libraries
containing isomers may be screened to determine, for example, if
only one isomer (e.g. an enantiomer or diastereomer) is binding to
the target receptor, or if the isomers have different affinities
for the target receptor. In this regard, if the isomers have
different affinities for the target receptor, a different break
through time will be observed for each isomer.
[0124] The compound libraries employed in this invention will
typically contain a plurality of members or putative ligands. When
a indicator compound is employed, the compound library will
preferably contain less than about 50,000 members, more preferably,
the compound library will contain less than about 10,000 members.
When an indicator compound is not employed, the compound library
will preferably contain less than about 10,000 members; more
preferably, from 1 to about 1,000 members; and still more
preferably, from about 5 to about 100 members.
[0125] The present apparatus is useful for analyzing the affinity
of members of a compound library for any target receptor or domain
which binds or complexes with a ligand. For example, the target
receptor may be selected from, but is not limited to, proteins,
including recombinant proteins, glycoproteins, glycosaminoglycans,
proteoglycans, integrins, enzymes, lectins, selectins,
cell-adhesion molecules, toxins, bacterial pili, transport
proteins, receptors involved in signal transduction or
hormone-binding, hormones, antibodies, major histocompatability
complexes, immunoglobulin superfamilies, cadherins, DNA or DNA
fragments, RNA and RNA fragments, whole cells, cell fragments,
tissues, bacteria, fungi, viruses, parasites, preons, and synthetic
analogs or derivatives of any of the above. If desired, more than
one target receptor may be employed when screening a compound
library using the methods of this invention.
[0126] When employing the apparatus of this invention, the target
receptor (or a ligand) is optionally bound or coupled to a solid
support. Preferably, the target receptor is covalently bound or
coupled to the solid support. However, in some cases, such as when
whole cells or organisms are employed as the target receptor, the
cells or organisms may be contained within the column by using, for
example, a porous frit or membrane at the outflow end of the
column. Supports for receptors are well-known in the art and many
are commercially available. Any such conventional support may be
used in this invention. Representative supports include, by way of
illustration, polymeric (resin) beads, polymeric gels, glass beads,
silica chips and capillaries, agarose, diatomaceous earths, pulp,
and the like. When silica capillaries are used as the solid
support, the target receptor is bound directly to the walls of the
column. Preferred solid supports for use in this invention are
those having minimal non-specific binding properties. A preferred
solid support is derivatized porous polystyrene-divinylbenzene
polymer beads, such as POROS beads (available from Perseptive
Biosystems, Framingham, Mass.). A particularly preferred solid
support is silica particles, such as controlled pore glass
(available from CPG Inc., Lincoln Park, N.J.).
[0127] The target receptor (or ligand) can be bound or coupled to
the support using any art-recognized procedure. For example, the
target receptor can be bound using direct immobilization techniques
(i.e., covalent binding via a sulfhydryl, amino or carboxyl group
and the like), covalent binding through a linking or spacer arm,
biotin-avidin binding, biotin-streptavidin binding, antibody
binding such as antibody-protein A binding, GST-glutathione
binding, ion exchange absorption, hydrophobic interaction,
expression of the target receptor as a recombinant protein fused to
maltose binding protein, fusion of the target receptor with a
peptide which binds selectively to an affinity column, and the
like. Such methods are well-known in the art and kits for
practicing many of these methods are commercially available. See,
for example, Stammers et al.., FEBS Lett. 1991, 283, 298-302;
Herman et al.., Anal. Biochemistry 1986, 156, 48; Smith et al.,
FEBS Lett. 1987, 215, 305; Kilmartin et al., J. Cell. Biol. 1982,
93, 576-582; Skinner et al., J. Biol. Chem. 1991, 266, 14163-14166;
Hopp et al., Bio/Technology 1988, 6, 1204-1210; H. M. Sassenfeld,
TIBTECH 1990, 8, 88-93; Hanke et al., J. General Virology 1992, 73,
654-660; Ellison et al., J. Biol. Chem. 1991, 267, 21150-21157; U.
K. Pati, Gene 1992, 114, 285-288; Wadzinski et al., J. Biol Chem.
1992, 267, 16883-16888; Field et al., Mol. Cell. Biol. 1988, 8,
2159-2165; Gerard et al., Biochemistry 1990, 29, 9274-9281;
Ausselbergs et al., Fibrinolysis 1993, 7, 1-13; Hopp et al.,
Biotechnology 1988, 6, 1205-1210; Blanar et al., Science 1992, 256,
1014-1018; Lin et al., J. Org. Chem. 1991, 56, 6850-6856; Zastrow
et al., J. Biol. Chem. 1992, 267, 3530-3538; Lim et al., J.
Infectious Disease 1990, 162, 1263-1269; Goldstein et al., Virology
1992, 190, 889-893; and the articles in IBI FLAG Epitope Vol. 1:
No. 1, September 1992; and references cited therein. Each of these
references is incorporated herein by reference in its entirety.
[0128] In a preferred embodiment of this invention, the target
receptor is bound or coupled to the solid support using
biotin-avidin, biotin-streptavidin or a related-type binding. In
this procedure, the target receptor is typically biotinylated with
a biotin reagent containing a spacer arm. The biotinylated target
receptor is then contacted with an avidin-containing solid support.
The resulting biotin-avidin complex binds the target receptor to
the solid support.
[0129] Procedures for biotinylating biomolecules are well-known in
the art and various biotin reagents are commercially available.
See, for example, E. A. Bayer et al., Meth. Enzymol. 1990, 184, 51;
U. Bickel et al., Bioconj. Chem. 1995, 6, 211; H. Hagiwara et al.,
J. Chromatog. 1992, 597, 331; "Avidin-Biotin Chemistry Handbook"
(available from Pierce, Rockford, Ill., Catalog Item No. 15055) and
references cited therein. A preferred biotin reagent is
NHS-LC-biotin (available from Pierce). The extent of biotin
incorporation using such reagents can be monitored by, for example,
matrix-assisted laser desorption/ionization as described in D. C.
Schriemer and L. Li, Anal. Chem. 1996, 68, 3382-3387, or by other
art-recognized methods as described in the "Avidin-Biotin Chemistry
Handbook" (Pierce). Preferably, an average of about 1 to about 50
biotins are incorporated per target receptor, more preferably about
1 to about 10 biotins per target receptor.
[0130] The biotinylated target receptor is typically coupled with
an avidin- or streptavidin-containing solid support or related
material. Such supports are commercially available or can be
prepared by art-recognized procedures. Preferred avidin-containing
supports include Ultralink-immobilized avidin (available from
Pierce) and POROS 20 immobilized streptavidin (available from
Perseptive Biosystems). The biotinylated target receptor is
typically coupled with the avidin-containing support by contacting
the receptor with the support in a suitable buffer, such as
phosphate buffered saline (pH 7), for about 0.5 to 4 hours at a
temperature ranging from about 4.degree. C. to about 37.degree. C.
Preferably, after coupling the biotinylated target receptor to the
avidin-containing support, any remaining avidin binding sites on
the support are blocked by contacting the solid support with an
excess of free biotin.
[0131] The target receptor may be bound or coupled to the solid
support either prior to or after introducing the solid support
material into a column. For example, the biotinylated target
receptor may be contacted or incubated with the avidin- or
streptavidin-containing solid support and the resulting solid
support containing the target receptor subsequently introduced into
a column. Alternatively, the avidin- or streptavidin-containing
solid support can be first introduced into the column and the
biotinylated target receptor then cycled through the column to form
the solid support containing the target receptor in the column.
Either of these methods may also be used with any of the other
previously mentioned procedures for coupling the target receptor to
the solid support.
[0132] When more than one target receptor is employed, each target
receptor can be bound to the same solid support using the
procedures described herein. Alternatively, each target receptor
can be bound to a separate solid support and the solid support
materials containing the target receptors subsequently homogenized
and packed into the column.
[0133] The solid support material may be introduced into the column
using any conventional procedure. Typically, the solid support is
slurried in a suitable diluent and the resulting slurry is pressure
packed or pumped into the column. Suitable diluents include, by way
of example, buffers such as phosphate buffered saline (PBS)
solutions, preferably containing a preservative such as sodium
azide, and the like.
[0134] Generally, the activity of the target receptor will
determine the size of the column employed in this invention, i.e.,
a smaller column volume may be employed when the target receptor
has more activity per unit column volume. Typically, the column
employed in this invention will have an internal diameter (i.d.)
ranging from about 10 .mu.m to about 4.6 mm. Preferably, the
internal diameter of the column will be in the range of from about
100 .mu.m to about 250 .mu.m. The column will typically range in
length from about 1 cm to about 30 cm, preferably from about 2 cm
to about 20 cm. Preferably, the column will have from about 1 fmol
to about 10 nmol of target receptor active sites per column; more
preferably, from about 0.1 pmol to about 10 nmol of target receptor
active sites per column; still more preferably, from about 0.1 pmol
to about 100 pmol of target receptor active sites per column.
[0135] If an indicator compound is employed, the length of the
column and its i.d. will also depend upon the K.sub.d of the
indicator compound (i.e., a smaller column may be used when the
indicator has a higher affinity for the target receptor).
Preferably, when an indicator is employed, the column length and
i.d. are selected so that the indicator compound elutes a
measurable quantity after the void volume.
[0136] The body of the column employed in this invention may be
comprised of any conventional column body material including, by
way of illustration, poly(ether ether ketone) (PEEK), fused silica,
silicon microchips, stainless steel, nylon, polyethylene,
polytetrafluoroethylene (Teflon) and the like. Preferably, the
column body is comprised of poly(ether ether ketone).
[0137] Alternatively, the column may be open-faced, such as in
thin-layer chromatography (TLC) plate configuration or a gel plate.
In this embodiment, the effluent can be analyzed using the
electrospray techniques described herein. Alternatively, the plate
or plate-like configuration can be subjected to matrix-assisted
laser desorption/ionization (MALDI) analysis at any stage of the
chromatography to provide a molecular weight analysis for a
plurality of positions on the plate.
[0138] After the solid support containing the target receptor is
introduced or formed in the column, the column is typically flushed
with a suitable diluent to remove any unbound target receptor or
impurities. Suitable diluents for flushing the column include, for
example, phosphate buffered saline, TRIS buffers and the like. If
desired, a detergent may also be included in the buffer to
facilitate removal of unbound target receptor or impurities.
[0139] After the column is flushed, the column is typically
equilibrated with a buffer suitable for frontal chromatography and
compatible with mass spectrometry. Volatile buffers are generally
preferred for use with mass spectrometry. For frontal
chromatography, a buffer is typically selected to promote
receptor-ligand interaction. Suitable buffers for use in FC-MS
include, by way of example, ammonium acetate, ammonium formate and
the like.
[0140] Following equilibration of the column, the compound library
is then applied to the column under frontal chromatography
conditions. Typically, when applied to the column, the compound
library comprises a solution of the library members or putative
ligands in a suitable diluent. Typically, the diluent is the buffer
solution used to equilibrate the column. Generally, the
concentration of the library members in the diluent will range from
about 1 pM to about 50 .mu.M. Preferably, the concentration of
library members ranges from about 1 nM to about 10 .mu.M.
[0141] Procedures for conducting frontal chromatography are
well-known in the art. See, for example, K.-I. Kasai et al.,
Journal of Chromatography 1986, 376, 33-47; D. S. Hage et al.,
Journal of Chromatography B, 1997, 669, 449-525 and references
cited therein. The disclosures of these references are incorporated
herein by reference in their entirety. Typically, the compound
library is continuously applied or infused into the column
containing the target receptor. Under these conditions, the target
receptor is continuously contacted or challenged with each of the
members of the compound library. The column is driven to dynamic
equilibrium by continuously applying the compound library to the
column. Library members having different binding constants to the
target receptor display different break through times or hold-up
volumes on the column, i.e., those members having a higher affinity
for the target ligand have a longer break through time on the
column or a larger hold-up volume until they begin to elute from or
break-though the column at their initial infusion concentration.
Unlike zonal chromatographic methods, no physical separation of the
library members is achieved using frontal chromatography. Suitable
methods for conducting FC-MS are described in U.S. patent
application No. ________, filed on Dec. 28, 1998 (which application
is a continuation of U.S. Ser. No. 09/070,131, filed Apr. 29, 1998,
now abandoned) and in U.S. Pat. No. ______, filed on even date
herewith, as Attorney Docket No. 026579-250 and entitled "Methods
for Screening Compound Libraries," the disclosure of which are
incorporated herein by reference in their entirety
[0142] During the frontal chromatography, the column is typically
at a temperature in range from about 0.degree. C. to about
90.degree. C.; preferably from about 4.degree. C. to about
60.degree. C.; more preferably from about 20.degree. C. to about 40
.degree. C.
[0143] When a ligand has a very slow on-rate, it may be desirable
to conduct the column equilibrium over an extended period of time.
The column can be equilibrated by infusing the compound library
through the column for a period sufficient to allow the column to
reach equilibrium. For example, this can be achieved by increasing
the equilibration time, i.e., to about 0.25 to 24 hours; or by
reducing the flow rate to about 1% to 10% of the usual flow rate.
Alternatively, a sequence of stop-flow cycles may also be
conducted.
[0144] In the apparatus of this invention, a mass spectrometer is
coupled to the column to analyze the effluent. Mass spectrometry is
particularly useful in the present invention since it allows for
both detection and identification of the library members present in
the effluent. In this regard, mass spectrometry allows the eluting
members of the library to be identified based on their mass/charge
ratio.
[0145] Prior to analyzing the effluent from the column by mass
spectrometry, the effluent is optionally diluted with a
supplemental diluent or "make-up flow" and the combined flow is
directed into, for example, the electrospray mass spectrometer.
Typically, the supplemental diluent comprises a major amount of an
organic solvent and a minor amount of an aqueous buffer. The
organic solvent is selected so as to promote a stable and efficient
electrospray. Representative organic solvents suitable for use in
the supplemental diluent include, by way of example, acetonitrile,
methanol, isopropanol and the like. A preferred organic solvent is
acetonitrile. Typically, the amount of supplemental diluent
employed is adjusted so that the combined flow rate of the effluent
and the supplemental diluent is less than about 100 .mu.L/min.
Preferably, the combined flow rate entering the mass spectrometer
ranges from about 100 nL/min to about 20 .mu.L/min.
[0146] Methods for analyzing effluents using mass spectrometry are
well-known in the art. Any type of mass spectrometry which is
capable of directly or indirectly analyzing the components present
in a solution may be employed in this invention including, for
example, electrospray mass spectrometry (ES-MS), atmospheric
pressure chemical ionization (APCI), membrane introduction mass
spectrometry (MIMS), continuous flow fast atom bombardment
(cf-FAB), thermospray techniques, particle beam, moving belt
interfaces and the like. Electrospray mass spectrometry is
particularly preferred. Apparatus and techniques for conducting
electrospray mass spectrometric analysis are described, for
example, in S. J. Gaskell, "Electrospray: Principles and Practice",
J. Mass. Spectrom. 1997, 32, 677-688 and reference cited therein.
When the effluent is collected and optionally pre-treated prior to
mass spectral analysis, any of the above described ionization
methods may be used as well as MALDI, fast atom bombardment (FAB),
massive cluster impact, electron impact, chemical ionization,
secondary ion mass spec and field desorption ionization
techniques.
[0147] The mass spectrometer employed in the methods of this
invention may be of any type (i.e., scanning or dynamic) including,
by way of illustration, quadrupole, time of flight, ion trap, FTICR
and the like. Typically, the mass spectrometer parameters are set
to provide the highest sensitivity for the eluting compounds.
Generally, when an electrospray mass spectrometer is employed, such
adjustments will involve optimization of, for example, nebulizer
pressure, drying gas flow rate, ion transmission and electrospray
needle position. For example, the nebulizer pressure will typically
range from about 0 psi to about 60 psi; and the drying gas flow
rate will range from about 0 L/min to about 50 L/min. A total ion
chromatogram is typically measured and monitored in real-time. The
size of the column, the concentration of the compound library and
the flow rate will generally determine the run-time. Typical run
times range from about 1 min to about 60 min.
[0148] Upon completion of the frontal chromatography, the column is
optionally regenerated by washing with a large volume of the
binding buffer, with or without a competitive ligand. In this
regard, a particular advantage of the present method is that
denaturing of the target receptor is not required at any point in
the procedure. Accordingly, columns may be re-used many times
generally with no observable loss of activity or leaching of the
target receptor. Alternatively, since the methods of this invention
employ very small amounts of target receptor, the column may be
disposed of after a single use.
[0149] A representative apparatus for conducting the screening
methods of this invention is illustrated in FIG. 1. As shown in
FIG. 1, a first reservoir 1, containing a buffer solution, and a
second reservoir 2, containing a solution of a compound library in
a buffer, are connected via tubing 3 to valve 4. In FIG. 1,
reservoirs 1 and 2 are syringes although any similar reservoir may
be employed. Valve 4 allows the solutions from reservoirs 1 or 2 to
be directed into a waste container 5 or into the inflow end of
column 6. Column 6 contains the target receptor bound to a solid
phase support, the column wall or otherwise retained within the
column. The outflow end of column 6 is connected to a mixing tee 7,
which is also connected to reservoir 8, containing a supplemental
diluent, via tubing 9. The effluent from column 6 is mixed with the
supplemental diluent from reservoir 8 in mixing tee 7 and the
outflow is directed via tubing 10 to an electrospray mass
spectrometer 11. To control the flow from reservoirs 1, 2 and 8,
pressure is applied to plungers 12 via, for example, a pump. A
simpler manifestation would include just the column 6 connected to
the reservoir 2, with the outflow directed via tubing 10 to an
electrospray mass spectrometer. Alternatively, a configuration
involving an HPLC pump and a valve with an oversized injection loop
for the library solution could be used, such an apparatus is
described, for example, in E. Breklan et al., Biochem. 1996, 35,
12141-12145.
[0150] In another of its embodiments, the apparatus of this
invention can be used for screening a compound library to determine
if any member of the library has an affinity for a target receptor
that interferes with the binding of a pre-selected indicator
compound or a mixture of indicator compounds. In this embodiment,
the break through time of an indicator compound having a known
affinity for the target receptor is determined after the column has
been equilibrated with the compound library and compared to the
break through time for the indicator compound in the absence of the
compound library. If the indicator compound has a shorter break
through time after equilibration with the compound library, the
compound library contains one or more ligands having an overall
affinity for the target ligand which is higher than the indicator
compound. Since an indicator compound can be selected having a
relatively short break through time on the column, a significant
advantage of this embodiment is that compound libraries can be
rapidly screened, e.g., in less than 5 minutes, to identify those
libraries having a pre-determined minimum level of affinity for the
target receptor. When a library is identified as having the
pre-determined minimum level of affinity for the target receptor,
the library can be further analyzed using FC-MS to identify the
ligands binding to the target receptor.
[0151] One advantage of using an indicator compound is that the
screening time for each library is significantly reduced since only
the indicator compound needs to be monitored relative to a void
marker compound. Additionally, since the indicator compound binds
to the target receptor at the active site of interest, a change in
the break through time for the indicator reflects an affective
interaction of a member (or members) of the library with the target
receptor. This interaction includes, by way of example, binding at
the active site, and binding at a different , non-overlapping site
that affects the ability of the target receptor to bind the
indicator compound. This method is particularly advantageous in
that nonspecific binding to the target receptor that does not alter
the active site will not cause a shift in the break through time
for the indicator compound. Accordingly, non-specific binding of
the library to the target receptor does not provide false
leads.
[0152] The indicator compound used in this embodiment of the
invention is typically selected so as to have a relatively weak
affinity for the target receptor. This permits the indicator
compound to rapidly elute or break through the column, thus
shortening the period of time necessary to monitor the effluent. An
indicator compound having a break through time on the column less
than about 5 minutes in the absence of the compound library is
preferred. Alternatively, an indicator having a strong affinity for
the target receptor may be used thereby allowing smaller columns to
be employed. When an indicator compound having a strong affinity is
used, the compound library will typically be applied to the column
at a higher concentration. The break through time for the indicator
compound on the column in the absence of the compound library is
determined using the FC-MS procedures described herein. The
affinity of the indicator compound for the target receptor can be
determined using conventional techniques, such as microcalorimetry
and the like; or by using the FC-MS methods of this invention.
Preferably, the indicator compound will also have a unique mass in
comparison to the members of the compound library so that the
indicator compound can be unambiguously identified by mass
spectrometry. Generally, when using an indicator compound and a
quadrupole mass spectrometer, only the m/z of the indicator
compound and the compounds representing the void volume are
monitored to provide for a greater signal to noise ratio.
[0153] Representative examples of indicator compounds suitable for
use with specific target receptors include, by way of illustration,
.alpha.Abe(1.fwdarw.3).alpha.Tal-OCH.sub.3 (K.sub.d =0.2 mM) for
use with a monoclonal antibody that recognizes the
3,6-dideoxy-D-galactose (abequose) epitope in Salmonella paratyphi
B O-antigens; phytic acid (K.sub.d=1 .mu.M) for use with
L-selectin, and the like. Additionally, more than one indicator
compound may be employed. The indicator may also be coupled or
conjugated to another molecule containing an atom, isotope or
molecular fragment which facilitates its detection. For example,
the indicator compound can be conjugated to polyethylene glycols
(PEGs) so that the mass spectra would contain peaks differing by 44
units thereby facilitating detection of the of indicator
compound.
[0154] The break through time for the indicator compound is
typically measured relative to a void marker compound. The void
marker compound is a compound which elutes from the column at the
void volume. Preferably, the void marker compound is structurally
similar to the indicator compound, but has no affinity for the
target receptor of interest. In some cases, putative ligands in the
compound library which have no affinity for the target receptor may
serve as the void marker compounds.
[0155] When a functional target receptor, such as an enzyme, is
employed in this invention, the substrate for the functional target
receptor may be used as the indicator. By doing so, the loss of
function or inhibition of the target receptor can be monitored in
the presence of a compound library. In this embodiment, a first
indicator compound is selected which is a substrate for the
functional target receptor, i.e. the first indicator is a compound
which is capable of being chemically modified by the functional
target receptor to produce a second indicator compound. Using the
frontal chromatography procedures described herein, the first
indicator compound and the compound library to be analyzed are
applied to or infused into a column comprising the functional
target receptor. The effluent from the column is then monitored for
the presence and/or concentration of the first and/or the second
indicator compounds, i.e., the substrate and/or the reaction
product. An increase in the expected concentration of the first
indicator compound or a decrease in the expected concentration of
the second indicator compound (as determined by conducting the
frontal chromatography of the indicator compounds in the absence of
the compound library) indicates that the functional target receptor
is being inhibited by one or more members of the compound library.
When a compound library is identified as having an inhibitor
present in the library for the functional target receptor, the
library can be further analyzed using FC-MS to identify the ligands
binding to the target receptor.
[0156] When using an indicator compound is employed, the break
through time for the indicator compound is first determined by
applying the indicator compound and the void marker compound to the
column containing the target receptor under frontal chromatography
conditions. The column is then typically equilibrated or partially
equilibrated with the compound library to be screened. Generally,
the compound library is applied or infused into the column for a
time sufficient to allow all of the library members to break
through the column. The effluent during this period may be
presented to the mass spectrometer for analysis or may be collected
for recycling or disposal. Once the column has been equilibrated or
partially equilibrated with the compound library, a mixture
comprising the compound library, void marker compound and the
indicator compound is applied to or infused into the column using
the frontal chromatography procedures described herein. Preferably,
the indicator compound will be present in the mixture in a
concentration less than its K.sub.d value. Typically, the indicator
compound will be present in an amount ranging from about 1 nM to
about 10 .mu.M, more preferably from about 10 nM to about 1 .mu.M.
The effluent from the column is analyzed to determine the break
through time for the indicator compound in the presence of the
compound library and this time period is compared to the
pre-determined break through time for the indicator compound to
ascertain whether the compound library has an affinity for the
target receptor.
[0157] Alternatively, the indicator compound and the void marker
compound, without the compound library, can be applied or infused
into the column after equilibration or partial equilibration of the
column with the compound library. This technique allows very
strongly bound ligands or those with slow off rates to be
detected.
[0158] An indicator compound can also be useful in determining
whether the overall affinity of a compound library is due to the
presence of a plurality of weak binders or to one or more strong
binders. In this embodiment, a column equilibration procedure is
initiated by infusion of the compound library as described herein.
The indicator compound, in the presence of the library and the
necessary void markers, is then passed through the column during
the initial stages of the equilibrium process (typically at about 1
to about 5 minutes) and a first break through time for the
indicator compound is determined. The flow of compound library
without the indicator is then reestablished through the column.
After a period of time, the indicator/library/void marker solution
is again passed through the column and a second break through time
is determined. When the same indicator compound is used through
this procedure, the intervening time period between each
application of the indicator compound can be as short as the time
necessary to wash off the indicator compound, and as long as the
total equilibration time (e.g., 1 min to 60 minutes, respectively).
This cycle can be repeated any number of times. In this method, the
discrimination between weak and strong binders occurs because a
weak ligand will reach equilibrium on the column sooner than a
strong one. Monitoring of the column activity during the
equilibration process is illustrated in FIG. 11 which is a graph of
the reduction of column activity as a function of time for two
different libraries: one containing many weak binders and one
containing strong binders. Even though the same overall reduction
in column activity is achieved (as measured by the indicator
compound), the rate of reduction is slower for the strong ligands
compared to the weak ligands.
[0159] An alternative method for distinguishing between a plurality
of weak binders and one or more strong binders in a compound
library is illustrated in FIG. 10. In this embodiment, an indicator
compound (V.sub.n-1 in FIG. 10) is first selected having an
affinity for the target receptor which is weaker than the putative
ligands of interest (V.sub.n) but stronger than those not of
interest (V.sub.1,max). A mixture comprising the compound library,
the void marker compound and the indicator compound at a
pre-determined initial concentration and signal intensity is then
applied or infused into a column comprising the target receptor
under frontal chromatography conditions to provide an effluent. In
this embodiment, the column is not pre-equilibrated with the
compound library. Preferably, the concentration of the indicator
compound in the mixture is greater than or equal to its
dissociation constant for the target receptor. The effluent from
the column is then analyzed by mass spectrometry to determine a
break through time for the indicator compound and its signal
intensity. In the presence of a plurality of weak binders (i.e.,
weaker than the indicator compound), the break through time for the
indicator will be less than its break through time in the absence
of the compound library and the basic shape of the curve will be
unchanged. In the presence of one or more ligands having an
affinity for the target receptor greater than the indicator
compound, the break through time will also be less than the break
through time for the indicator compound in the absence of the
compound library, but the shape of the curve will also display a
"roll up" effect as illustrated in FIG. 10. This "roll up" effect
is due to the removal of bound indicator compound by the stronger
ligand(s). Thus, for a short period of time, the concentration of
the indicator compound is higher than its infusion concentration
until the stronger ligand(s) breaks through. The amount of bound
indicator compound removed is dependent upon the K.sub.d value and
the concentration of the stronger ligand(s) present in the library.
Weaker ligands do not exert this "roll up" effect because they
propagate through the column more quickly than the indicator
compound. Detection or measurement of this "roll up" or bump in the
break through curve (typically measured as a change in signal
intensity) indicates the presence of ligands in the compound
library having an affinity for the target receptor greater than the
indicator compound. The detection or measurement of a "roll up"
effect may also be used when screening target receptors for
affinity to an immobilized ligand(s).
[0160] In addition to detecting the indicator compound using mass
spectrometry, other methods of detection may also be employed. Any
detection method that can measure the indicator compound over the
background signal of the library compounds can be used. For
example, an indicator compound can be detected in the effluent from
the column using, by way of example, fluorescence, infra-red
absorption, UV-visible absorption, nuclear magnetic resonance
(NMR), atomic spectroscopy (i.e., atomic adsorption spectroscopy
(AAS), inductively coupled plasma-optical emission spectroscopy
(ICP-OES), etc.), flow cytometry, electrochemical detection and the
like. Procedures and apparatus for detecting compounds using such
methods are well-known in the art and any conventional procedures
and apparatus may be used.
[0161] The apparatus of this invention allow a plurality of FC-MS
analyses to be conducted simultaneously using a single mass
spectrometer to intermittently monitor each column. Unlike "capture
and release" methods which typically provide an elution peak or
"spike" for each ligand, FC-MS does not require constant effluent
monitoring because once a library member breaks through the column,
that member is continuously present in the effluent and can be
detected by the mass spectrometer. Therefore, a plurality of FC-MS
analyses can be conducted simultaneously using a single mass
spectrometer to intermittently monitor each column. For example,
using this invention, at least about 100 columns can be conducted
simultaneously.
[0162] When employing multiple columns, each column is typically
monitored for a brief period of time before switching to the next
column. For example, with a quadrupole mass spectrometer, each
column is typically monitored sequentially for a period of about
0.5 seconds to about 10 seconds, preferably for about 1 second to
about 5 seconds, before switching to the next column. The effluent
from each column is analyzed as described herein using mass
spectrometry. Generally, a single data file is used to collect all
of the data from the multiple column thereby generating a composite
total ion chromatogram. Subsequently, separate total ion
chromatograms for each column are recreated by synchronizing column
switching with mass spectrometry data acquisition.
[0163] In a preferred embodiment, each column will have an
individual electrospray needle for injection of the column's
effluent into the electrospray mass spectrometer. Any geometric
arrangement of multiple electrospray needles that allows for fast
and repetitive sequences of needle advancement may be employed. A
suitable apparatus for the injection of multiple effluents into an
electrospray mass spectrometer is described in U.S. patent
application No. 09/069,656, filed Apr. 29, 1998, and U.S. Pat. No.
______, filed on even date herewith, as Attorney Docket No.
026579-260 and entitled "Device for Delivery of Multiple Liquid
Sample Streams to a Mass Spectrometer," the disclosures of which
are incorporated herein in their entirety. Alternatively, a linear
moving row of electrospray needles (sprayers) and the like may be
employed. See, for example, Q. Xue et al., Anal. Chem. 1997, 69,
426-430 and references cited therein, the disclosed of which is
incorporated herein by reference in its entirety.
[0164] A representative apparatus for screening compound libraries
using a plurality of columns is illustrated in FIG. 2. As shown in
FIG. 2, each of a plurality of columns 13 is connected via tubing
14 and tee 15 to a first reservoir 16, containing a solution of a
compound library in a binding buffer, and a second reservoir 17,
containing the binding buffer. In FIG. 2, reservoirs 16 and 17 are
syringes although any similar reservoir may be employed. Each
column 13 contains a target receptor bound to a solid phase
support. The buffer solution in reservoir 17 is used to wash column
13 before or after introduction of the compound library. The
outflow end of each column 13 is connected to a mixing tee 18,
which is also connected to reservoir 19, containing a supplemental
diluent, via tubing 20. The effluent from each column 13 is mixed
with the supplemental diluent from reservoir 19 in mixing tees 18
and the outflow is directed via tubing 20 and valves 21 into an
electrospray mass spectrometer 22, via an electronically-actuated
multi-port selection valve 23, or into waste/recovery containers
24. To control the flow from reservoirs 16, 17 and 19, pressure is
applied to plungers 25 via, for example, pumps.
[0165] Alternatively, in another embodiment illustrated in FIG. 3,
the outflow from mixing tees 18 may be directed via tubing 20 into
individual electrospray needles 26 for mass spectrometer
analysis.
[0166] When using an indicator compound, sequential runs of
multiple columns may be advantageous since this allows the
retention time for the indicator compound to be more accurately
determined. Parallel infusion of the indicator through a plurality
of columns is feasible provided an apparatus is used with a
suitable high sampling rate (e.g., allowing for a minimum of five
mass spectral measurements on the break through curve of the
indicator for each of the columns. Such an apparatus is described
in U.S. patent application No. 09/069,656, filed Apr. 29, 1998, and
U.S. Pat. No. ______, filed on even date herewith, as Attorney
Docket No. 026579-260.
[0167] A representative apparatus for sequentially screening
compound libraries with a indicator compound using a plurality of
columns is illustrated in FIGS. 2, 3 and 4. The apparatus shown in
FIGS. 2 and 3 are preferred and are employed as described herein
for FC/MS. However, reservoir 16 optionally contains a solution of
the compound library plus the indicator compound and void marker
compounds in a binding buffer, while reservoir 17 optionally
contains a solution of only the compound library in the binding
buffer. Alternatively, the apparatus illustrated in FIG. 4 can be
used. As shown in FIG. 4, a plurality of reservoirs 27 (e.g.,
syringes) are held in place with clamp 38. Each reservoir 27
contains a mixture of a compound library and an indicator compound
in a suitable diluent (or, alternatively, simply the indicator).
The end of each reservoir 27 is connected via tubing 29 to the
inflow end of a column 30 containing the target receptor bound to a
solid phase support. The outflow end of each column 30 is connected
via tubing 31 to an electronically-actuated multiport stream
selection valve 32 which controls the flow of the effluent from
columns 30. Using valve 32, the effluent from the columns may be
directed into a waste container 33, via tubing 34, or into mixing
tee 35, via tubing 36. Mixing tee 35 is also connected to reservoir
36, containing a supplemental diluent, via tubing 37. The effluent
from each column 30 is mixed with the supplemental diluent from
reservoir 36 in mixing tee 35 and the outflow is directed via
tubing 38 into an electrospray mass spectrometer 39. To control the
flow from the reservoirs 27 into columns 30, a stand-off block 40
may be employed. When pressure is applied to stand-off block 40
via, for example, a pump, the plunger 41 of each reservoir 27 is
individually depressed in sequence thereby infusing the contents of
the reservoir through tubing 29 into the corresponding column 30.
The effluent emerging from each column 30 is sequentially directed
into mass spectrometer 39 for analysis.
[0168] The apparatus of this invention also permit the absolute
affinity or dissociation constant, K.sub.d, for certain individual
members of a compound library to be readily determined. In this
regard, ligands having an affinity for the target receptor break
through the column at volumes (i.e., break through times) related
to their concentrations and K.sub.d values, according to the
following equation: 1 V x - V 0 = B t [ X ] 0 + ( K d ) x
[0169] where B.sub.t represents the dynamic binding capacity of the
column; [X].sub.0 is the infusion concentration of the ligand in
the compound library; K.sub.d is the dissociation constant for the
ligand; V.sub.0 is the void volume; and V.sub.x represents the
volume at the mid-point of the front corresponding to the break
through of the ligand. This simple equation indicates that, once
B.sub.t and the concentration of the ligand are known, the
dissociation constant of a ligand can be determined from a single
measurement of its V.sub.x-V.sub.0. This equation strictly applies
only in the case of a single ligand. In many cases, however, this
equation or a modification of it can be applied to multiple ligands
as well.
[0170] In order to determine B.sub.t, a representative compound,
e.g., compound X, is infused through the column at various
concentrations and the corresponding V.sub.x-V.sub.0 values
measured. A plot of ([X](V-V.sub.0)).sup.-1 versus [X].sup.-1 is
generated, where the y-intercept indicates the dynamic binding
capacity of the column (B.sub.t) (analogous to a Lineweaver-Burk
plot).
[0171] Once the dynamic binding capacity of the column has been
determined, the dissociation constants for individual members of
the compound library can be determined from a single FC-MS run. For
example, the K.sub.d for compounds where [X]<<(K.sub.d).sub.x
is determined simply from B.sub.t/(V-V.sub.0). For those members of
the library with a low dissociation constant, knowledge of their
concentration or infusion of the compound library at higher
dilution is required to determine K.sub.d.
[0172] The following examples are offered to illustrate this
invention and are not to be construed in any way as limiting the
scope of this invention. Unless otherwise stated, all temperatures
are in degrees Celsius.
EXAMPLES
[0173] In the examples below, the following abbreviations have the
following meanings. If an abbreviation is not defined, it has its
generally accepted meaning.
[0174] B.sub.t=dynamic binding capacity
[0175] .degree.C.=degrees Celsius
[0176] cm=centimeter
[0177] eq.=equivalents
[0178] FAB=fast atom bombardment
[0179] FC=frontal chromatography
[0180] g=grams
[0181] K.sub.d=dissociation constant
[0182] L=liter
[0183] MALDI=matrix-assisted laser desorption/ionization
[0184] meq.=milliequivalent
[0185] mg=milligram
[0186] mL=milliliter
[0187] mM=millimolar
[0188] mmol=millimole
[0189] MS=mass spectrometry
[0190] m/z=mass charge ratio
[0191] N=normal
[0192] PBS=phosphate buffered saline
[0193] PEEK=poly(ether ether ketone)
[0194] pmol=picomole
[0195] TIC=total ion chromatogram
[0196] .mu.g=micrograms
[0197] .mu.L=microliter
[0198] .mu.m=micrometer
[0199] .mu.M=micromolar
[0200] V.sub.0=void volume
Example 1
Screening of an Oligosaccharide Library Using FC-MS
[0201] In this example, a compound library containing a mixture of
six oligosaccharides was screened using frontal chromatography in
combination with an electrospray mass spectrometer to determine the
relative affinity of the oligosaccharides for a monoclonal antibody
that recognizes the 3,6-dideoxy-D-galactose (abequose) epitope in
Salmonella paratyphi B O-antigens.
[0202] The compound library consisted of the following six
oligosaccharides: .alpha.GalNAc(1.fwdarw.3).beta.Gal-OGr (compound
1); .alpha.Gal(1.fwdarw.3)[.alpha.Fuc(1.fwdarw.2)].beta.Gal-OGr
(compound 2);
.alpha.Man(1.fwdarw.3)[.alpha.Man(1.fwdarw.6)]PMan-OGr (compound
3); .alpha.Abe(1.fwdarw.3).alpha.Tal-OCH.sub.3 (compound 4);
.alpha.Gal(1.fwdarw.2)[.alpha.Abe(1.fwdarw.3)].alpha.Man-OCH.sub.3
(compound 5); and
.alpha.Glc(1.fwdarw.4).beta.Glc(1.fwdarw.4).alpha.Gal(1-
.fwdarw.2)-[.alpha.Abe(1.fwdarw.3)].alpha.Man(1.fwdarw.3).alpha.Glc(1.fwda-
rw.4).beta.Glc-OCH.sub.3 (compound 6), wherein
Gr=O(CH.sub.2).sub.8CO.sub.- 2CH.sub.3. Compound 1-3 were obtained
using the procedures described in U.S. Pat. No. 4,362,720 to R. U.
Lemieux et al., issued Dec. 7, 1987; U.S. Pat. No. 4,137,401 to R.
U. Lemieux et al, issued Jan. 30, 1979; and K. J. Kaur et al., "Use
of N-Acetylglucosaminyltransferases I and II in the Preparative
Synthesis of Oligosaccharides", Carbohydr. Res. 1991, 210, 145-153;
respectively, the disclosures of which are incorporated herein by
reference in their entirety. Compounds 4-6 were obtained using the
procedures described in D. R. Bundle et al., "Modulation of
Antibody Affinity by Synthetic Modifications of the Most Exposed
Pyranose Residue of A Trisaccharide Epitope", Bioorg. Med. Chem.
1994, 2, 1221-1229, the disclosure of which is incorporated herein
by reference in its entirety. Compounds 1-3 are known to have no
specificity for the antibody. On the other hand, compounds 4-6
contain the minimal requirement for recognition (abequose) and span
a range of affinity for the antibody. The K.sub.d values for
compounds 4-6, as determined by titration microcalorimetry, are
shown in Table 1 below.
[0203] The monoclonal antibody used in this experiment was produced
as described in D. R. Bundle et al, "Molecular Recognition of a
Salmonella Trisaccharide Epitope by Monoclonal Antibody Se155.4"
Biochem. 1994, 33, 5172-5182. The antibody (0.5 mg) was
biotinylated with a biotin reagent containing a long-chain spacer
arm (NHS-LC-biotin, Pierce). The extent of biotin incorporation was
monitored by matrix-assisted laser desorption/ionization and the
reaction was terminated at 14 biotins/IgG (average). The
biotinylated antibody was then coupled to a beaded support by
incubating the antibody with 25 .mu.L of Ultralink immobilized
avidin (Pierce, Cat. No. 53119) in bicarbonate buffer (pH 8.5) for
1 hour. The beads were then thoroughly washed with the bicarbonate
buffer. A UV quantitation indicated an immobilization of .about.45
.mu.g antibody/25 .mu.L beads was achieved. The beads were then
slurry-packed into a 500 .mu.m i.d. by 11.5 cm poly(ether ether
ketone) (PEEK) column body (.about.23 .mu.L column volume).
[0204] In this experiment, a mixing tee served a dual role as a
column end-fitting and mixing chamber for the column eluent and
organic make-up flow. The column was then directly connected to an
electrospray mass spectrometer (Hewlett-Packard series 1100 MSD,
single quadrupole).
[0205] For operation in frontal chromatography mode, the column was
first flushed with ammonium acetate buffer (NH.sub.4OAc, 2 mM, pH
6.7). After flushing , the flow was switched to a second solution
containing a mixture of the six oligosaccharides in ammonium
acetate buffer, each present at 1 .mu.M. All solutions were infused
concurrently with a multi-syringe pump (PHD 200, Harvard Apparatus)
at a flow rate of 8 .mu.L/min/syringe (1 cc syringes). A Rheodyne
valve (Model 9725) was used for flow switching. The column effluent
combined with the make-up flow (10% 2 mM NH.sub.4OAc buffer in
acetonitrile) in the tee to provide a flow rate of 16 .mu.L/min
into the mass spectrometer.
[0206] For the analysis of this mixture, the spectrometer was
scanned from m/z 100-1500. Data was collected in scan mode with
positive ion detection. A total ion chromatogram (TIC) was
constructed from a 50 minute run time as shown in FIG. 5A. This
represented the consumption of only 400 pmol of each
oligosaccharide. Peaks at specific m/z values were then identified
through the analysis of the mass spectra giving rise to the TIC and
selected ion chromatograms for all six compounds were reconstructed
from the TIC as shown in FIG. 5B. Compounds 1-3 break through the
column simultaneously as indicated by the solid line. Mass spectra
were then generated from time-slices of the TIC (at times I, II and
III) as shown in FIGS. 5C, 5D and 5E. These mass spectra chart the
progression of the various oligosaccharides through the column. A
spectrum representing the onset of compound 4 is not shown.
[0207] As discussed above, ligands having no affinity for the
target receptor break through at the void volume (V.sub.0), while
compounds having an affinity for the target ligand break through
later, at volumes relating to their concentrations and K.sub.d
values, according to the following equation: 2 V x - V 0 = B t [ X
] 0 + ( K d ) x
[0208] where B.sub.t represents the dynamic binding capacity of the
column; [X].sub.0 is the infusion concentration of the ligand in
the compound library; K.sub.d is the dissociation constant for the
ligand; V.sub.0 is the void volume; and V.sub.x represents the
volume at the mid-point of the front corresponding to the break
through of the ligand.
[0209] In order to determine B.sub.t, compound 5 was infused
through the column at various concentrations and the corresponding
V-V.sub.0 values measured. A plot of
([A].sub.0(V-V.sub.0)).sup.-1versus [A].sub.0.sup.-1 was generated,
where A is compound 5, as shown in FIG. 6. The y-intercept
indicated a B.sub.t of 520 pmol. Each antibody molecule contains
two binding sites, therefore this corresponds to an active capacity
of 260 pmol of protein (representing 93% of the total amount of
protein bound). The x-intercept indicated a K.sub.d of 11.2 .mu.M
for compound 5, which compares favorably with the value determined
by microcalorimetry as shown in Table 1.
[0210] Knowledge of the column capacity prior to the screening of a
mixture allows for the determination of dissociation constants from
a single frontal chromatogram. For compounds with
[X]<<(K.sub.d).sub.- x, the K.sub.d can be determined simply
from B.sub.t/(V-V.sub.0). For example, compound 4 was shown to have
a K.sub.d of 0.2 mM, as determined from the chromatogram of FIG.
5B. Compounds with low dissociation constants require either the
knowledge of their concentration or the infusion of the mixture at
higher dilution for the determination of K.sub.d. The K.sub.d of
compound 6, at a 1 .mu.M concentration, was determined from the
same chromatogram to be 1.5 .mu.M.
[0211] The column was regenerated offline by washing with a large
volume of binding buffer. The column used in this example was
subjected to over 150 runs with no observable loss of activity or
leaching of the antibody.
[0212] The results from this experiment are shown in Table 1.
1 TABLE 1 K.sub.d .+-. s (.mu.M).sup.2 Oligosaccharide Micro- FC/MS
No. Gr = O(CH.sub.2).sub.8CO.sub.2CH.- sub.3 (MNa).sup.+1 cal.sup.3
Ind..sup.4 Mix.sup.5 1 .alpha.GalNAc(1.fwdarw.3).beta.Gal--OGr
576.3 -- -- 2
.alpha.Gal(1.fwdarw.3)[.alpha.Fuc(1.fwdarw.2)].beta.GalO--Gr 681.3
-- -- 3
.alpha.Man(1.fwdarw.3)[.alpha.Man(1.fwdarw.6)].beta.Man--OGr 697.3
-- -- 4 .alpha.Abe(1.fwdarw.3).alpha.Tal--OCH.sub.3 347.0 190 185
.+-. 17 178 .+-. 23 5 .alpha.Gal(1.fwdarw.2)[.alpha.Abe(-
1.fwdarw.3)[.alpha.Man--OCH.sub.3 509.2 6.3 12.6 .+-. 1.3 10.2 .+-.
1.1 6
.alpha.Glc(1.fwdarw.4).beta.Glc(1.fwdarw.4).alpha.Gal(1.fwdarw.2-
)[.alpha.Abe 1157.4 0.88 1.79 .+-. 0.20 1.71 .+-. 0.16
(1.fwdarw.3)].alpha.Man(1.fwdarw.3).alpha.Glc(1.fwdarw.4).beta.Glc-
OCH.sub.3 .sup.1Monoisotopic molecular weight of the singly charged
sodium adduct. .sup.2Dissociation constant with the corresponding
standard deviation. .sup.3Microcalorimetry. .sup.4Values determined
from infusion of individual ligand, with Compounds 1-3.
.sup.5Values determined from the infusion of the six-compound
mixture.
[0213] The results in Table 1 demonstrate that the affinity of
various putative ligands in a compound library for a target
receptor can be determined relative to other putative ligands in
the library; and that the dissociation constant, K.sub.d, for
putative ligands and the target receptor can be determined. The
results further demonstrate that there is an acceptable correlation
between the literature K.sub.d values and those generated by FC-MS
procedures.
Example 2
Screening of an Oligosaccharide Library Using FC-MS and an
Indicator Compound
[0214] In this example, the use of an indicator compound to screen
a compound library is demonstrated. The antibody used in this
example was the same as that used in Example 1, i.e., a monoclonal
antibody that recognizes the 3,6-dideoxy-D-galactose (abequose)
epitope in Salmonella paratyphi B O-antigens. The column was also
essentially the same as the column in Example 1 and it was prepared
and operated as described therein.
[0215] In this experiment, three solutions were prepared. Solution
A contained the following four oligosaccharide in 2 mM NH.sub.4OAc:
.alpha.GalNAc(1.fwdarw.3).beta.Gal-OGr (compound 1);
.alpha.Gal(1.fwdarw.3)[.alpha.Fuc(1.fwdarw.2)].beta.Gal-OGr
(compound 2);
.alpha.Man(1.fwdarw.3)[.alpha.Man(1.fwdarw.6)].beta.Man-OGr
(compound 3); .alpha.Abe(1.fwdarw.3).alpha.Tal-OCH.sub.3 (compound
4), wherein Gr=O(CH.sub.2).sub.8CO.sub.2CH.sub.3. Solution B
contained
.alpha.Gal(1.fwdarw.2)[.alpha.Abe(1.fwdarw.3)].alpha.Man-OCH.sub.3
(compound 5) in 2 mM NH.sub.4OAc; and Solution C contained
compounds 1-5 in 2 mM NH.sub.4OAc. In all solutions, compounds 1, 2
and 3 were present at 1 .mu.M, compound 4 was present at 0.16
.mu.M, and compound 5 was present at 15 .mu.M. In this example,
compound 4 was used as the indicator compound and compound 5 was
used to represented a member of a compound library. The remaining
compounds were used to determine V.sub.0.
[0216] Solution A containing compounds 1-4 was infused into the
column as described in Example 1. A quadrupole mass spectrometer
was used to monitor the effluent. The mass spectrometer was
operated in selected ion monitoring (SIM) mode, on the
(M+Na).sup.+peak of each compound. FIG. 5A shows the selected ion
chromatograms generated from an infusion of compounds 1-4 (i.e.,
Solution A). The breakthrough volume for compound 4 was 3.0.+-.0.1
.mu.L. The column was regenerated by flushing with the binding
buffer (i.e., 2 mM NH.sub.4OAc) for about 10 min. at which time
essentially all traces of compound 4 were removed.
[0217] Using the apparatus of FIG. 1, Solution B (compound 5) and
Solution C (compounds 1-5) were loaded into separate syringes.
Solution B was infused through the column until dynamic equilibrium
for compound 5 was attained. At this point, the flow was switched
to the syringe carrying Solution C, and the selected ion
chromatograms of FIG. 7B were generated using the quadrupole mass
spectrometer. As shown in FIG. 7B, pre-equilibration of the column
with compound 5 leads to a measurable shift in the breakthrough
volume of the indicator compound 4 (to 1.1.+-.0.3 .mu.l). This is
consistent with the fact that compound 5 is a ligand having a
K.sub.d for the antibody lower than that of the indicator compound
4 (see Table 1 above). Therefore, by simply monitoring the
indicator compound, the fact that the representative library
contained a compound with a higher affinity for the target receptor
was readily apparent.
[0218] Note that while the indicator compound (compound 4) in this
experiment was added to a solution of the representative library
(compound 5), this will not always be necessary. In those
situations where the library (Solution B) contains a strongly
retained compound (i.e., low K.sub.d, or off-rate), Solution A can
be substituted for Solution C (i.e., the indicator does not need to
be mixed with the library).
Example 3
Screening of an Oligosaccharide Library Using FC-MS
[0219] In this example, a compound library containing a mixture of
four oligosaccharides was screened using frontal chromatography in
combination with an electrospray mass spectrometer to determine the
relative affinity of the oligosaccharides for cholera toxin B
subunit.
[0220] The compound library consisted of the following four
oligosaccharides: .alpha.GalNAc(1.fwdarw.3).beta.Gal-OGr (compound
1); .alpha.Gal(1.fwdarw.3)[.alpha.Fuc(1.fwdarw.2)].beta.Gal-OGr
(compound 2);
.alpha.Man(1.fwdarw.3)[.alpha.Man(1.fwdarw.6)].beta.Man-OGr
(compound 3); and GM.sub.1 oligosaccharide (compound 7, wherein
Gr=O(CH.sub.2).sub.8CO.- sub.2CH.sub.3. Compound 7, which is the
natural ligand for cholera toxin B subunit, was obtained using the
procedures described in A. Schon et al., "Thermodynamics of
Intersubunit Interactions in Cholera Toxin upon Binding to the
Oligosaccharide Portion of Its Cell Surface Receptor, Ganglioside
G.sub.M1" Biochem. 1989, 28, 5019-5024, the disclosure of which is
incorporated herein by reference in its entirety. Cholera toxin B
subunit was obtained from LIST Biochemicals, Campbell, Calif.
[0221] A column was prepared from a 12 cm section of 0.01" (250
.mu.m) i.d. PEEK tubing (column volume of about 6 .mu.L). The
column was packed with POROS 20 immobilized streptavidin particles
(available from Perseptive Biosystems, Framingham, Mass.).
[0222] Cholera toxin B subunit (a pentameric protein) was
biotinylated to provide about 1-2 biotins/monomer, as measured by
MALDI. A dilute solution of this biotinylated protein (4 .mu.M) was
infused through the pre-packed column such that the total amount of
cholera toxin B subunit bound was approximately 200 pmol after
washing (as determined by UV quantitation).
[0223] A solution containing compounds 1-3 and 7 was prepared. All
compounds were present at 2 .mu.M, in 2 mM NH.sub.4OAc (pH 6.9).
Using an apparatus similar to that shown in FIG. 1, the column was
first equilibrated with the binding buffer (2 mM NH.sub.4OAc). The
solution containing compounds 1-3 and 7 was then infused through
the column at 8 .mu.L/min. The effluent was combined with a typical
make-up flow (10% 2 mM NH.sub.4OAc in acetonitrile) and passed into
an electrospray single quadrupole mass spectrometer. Data was
collected in scan mode, with negative ion detection.
[0224] A total ion chromatogram was generated, followed by
reconstruction of selected ion chromatograms for each of compounds
1-3 and 7 as shown in FIG. 8. As illustrated in FIG. 8, compounds
1-3 broke through in the void volume of the system (.about.4
min.times.8 .mu.L/min=32 .mu.L) while compound 7 (GM.sub.1
oligosaccharide) broke through at .about.300 .mu.L. Thus, GM.sub.1
oligosaccharide (K.sub.d.congruent.100 nM) has a stronger affinity
for cholera toxin B subunit than compounds 1-3 which have little or
no affinity for cholera toxin B subunit.
[0225] A second mixture was then prepared in the binding buffer and
analyzed by FC-MS in a similar fashion. This mixture contained a
synthetically prepared GM.sub.1 analogue, i.e.,
.beta.Gal(1.fwdarw.3).bet-
a.GalNAc(1.fwdarw.)-OCH.sub.2CH.sub.2O-(.rarw.2).alpha.Neu5Ac,
(compound 8) in an impure form (i.e. containing unidentified
intermediates and reaction byproducts). Compound 8 was prepared by
the methods described in P. Fugedi et al, "A Novel Promoter for the
Efficient Construction of 1,2-trans Linkages in Glycoside
Synthesis, Using Thioglycosides as Glycosyl Donors" Carbohydr. Res.
1986, 149, C9-C12; A. Marra et al., Stereoselective Synthesis of
2-Thioglycosides of N-Acetylneuraminic Acid", Carbohydr. Res. 1989,
187, 35-42; and L. Lay et al., "Synthesis of the Propyl Glycoside
of the Trisaccharide .alpha.-L-Fucp-(1.fwdarw.2)-.be-
ta.-D-Galp-(1.fwdarw.3)-.beta.-D-GalpNAc. Components of a Tumor
Antigen Recognized by the Antibody Mbr1" Helv. Chim. Acta. 1994,
77, 509-514; the disclosures of which are incorporated herein by
reference in their entirety. The mixture was infused through the
column, and the mass spectrometer was set to operate in selected
ion monitoring mode, on negative ions representative of compounds 3
and 8. Selected ion chromatograms were generated for these ions as
shown in FIG. 9. FIG. 9 shows that compound 3 broke through in the
void volume (m/z 673.2). A more complex pattern was observed for
the ions with a mass/charge of 717.2 u. A certain fraction of these
ions also broke through in the void volume (.about.25%), while the
remaining 75% broke through significantly later (at about 11 min).
This two-front profile indicates an isobaric impurity exists at the
25% level, which does not bind to cholera toxin B subunit. Thus,
FC-MS is able to ascertain the presence of isobaric, non-binding
impurities. Reasonably accurate quantitation of these impurities
can also be achieved.
Example 4
Screening of a Compound Library Containing 100 Putative Ligands
Against a Human Enzyme
[0226] In this example, a compound library containing a mixture of
100 tripeptides was screened against immobilized human
.alpha.-thrombin using frontal chromatography in combination with
an electrospray mass spectrometer. The peptides were synthesized as
a mixture by established solid phase techniques and were purchased
from Alberta Peptide Institute (Edmonton, Alberta, Canada). This
set of peptides all have a common C-terminal amino acid (arginine),
while the remaining two positions are random and chosen from a set
of 10 amino acids (see FIG. 12). FIG. 12 also displays an
electrospray mass spectrum of the mixture. The spectrum was
collected from an infusion of a 50 .mu.M solution in 1:1
acetonitrile:ammonium acetate (2 mM, pH 7.2). Assuming equimolar
ratios of all peptides, this corresponds to 0.5 .mu.M per peptide.
This spectrum highlights a peak at m/z of 419.2. This peak
corresponds to two isomeric entries in the library: PfR and fPR,
where f refers to D-phenylalanine. The tripeptide fPR has been
identified in the literature as an inhibitor possessing a K.sub.d
value of approximately 1 .mu.M against human .alpha.-thrombin. An
experiment was conducted to determine if this ligand could be
detected when present in the full mixture as screened against a
thrombin column.
[0227] A column was prepared from a 5 cm section of 0.01" (250
.mu.m i.d.) PEEK tubing (column volume of about 2.5 .mu.L). The
column was packed with POROS 20 immobilized streptavidin particles
(available from Perseptive Biosystems, Framinghan, Mass.). Human
.alpha.-thrombin was purchased from Sigma Chemical Co., and
biotinylated with a reagent containing a long-chain spacer arm
(sulfo-NHS-LC-biotin, Pierce). The extent of biotin incorporation
was less than 5 biotins/thrombin, as monitored by matrix-assisted
laser desorption/ionization. A dilute solution of this biotinylated
protein (approximately 2 .mu.M) was infused through the pre-packed
column in the presence of 0.1% bovine serum albumin (w/v) such that
the total amount of immobilized thrombin was approximately 570 pmol
(as calculated by a determination of the reduced capacity of the
column for free biotin).
[0228] The BOC-protected fPR (BOC-fPR, also known as ligand) was
purchased from Calbiochem and infused through the column at a
concentration of 1 .mu.M in ammonium acetate solution (2 mM, pH
7.2), and at a flow rate of 8 .mu.L/min. FIG. 13 displays a
chromatogram reflecting three infusions/wash cycles of this peptide
through the column, in the presence of a void marker compound (a
non-binding trimannosyl compound). FIG. 13 shows the selected ion
chromatograms for each compound. The average V-V.sub.0 value was
determined to be 4.66 .mu.L. Based on this experiment, and the
infusion of this peptide at higher concentrations, the B.sub.t
value of the column was calculated to be approximately 145 pmol
(.about.25% active). The BOC-fPR was selected as the indicator
compound for the following work, with the trimannosyl compound the
corresponding void marker compound.
[0229] The library of peptides was then infused through the column
at a concentration of 1 .mu.M per peptide for approximately 30
minutes, whereupon the indicator compound and void market compound
(in the presence of the library) were infused. The same buffer and
flow rate conditions as above were used. FIG. 14 displays the
V-V.sub.0 value immediately before (14A) and immediately after
(14B) the 30 minute equilibration time. The drop in V-V.sub.0 as a
result of the 100 peptides indicates the loss of virtually all of
the binding activity of the protein.
[0230] To determine the nature of the compounds giving rise to the
indicator shift, the peptide library was infused through a fresh,
identically prepared column in FC/MS mode. The HP quadrupole
electrospray mass spectrometer was set to scan the mass range from
m/z 100 to 600 at a scan rate of approximately 1 sec/cycle. The
effluent was monitored in real time, and the experiment was stopped
at approximately 15 minutes. The results are displayed in FIGS. 15A
and 15B. FIG. 15A shows a featureless total ion chromatogram.
However, a generation of selected ion chromatograms from the peaks
representing the mixture components resulted in the identification
of m/z 419.2 as giving rise to the largest V-V.sub.0 shift (as
shown in FIG. 15B). Two breakthrough curves are evident in this
Figure, indicating the presence of at least two isomers. Based on a
knowledge of the mixture composition, this is consistent with the
presence of the isomers PfR (non-binding) and fPR (the ligand). A
fresh column was constructed through which a solution of just the
void marker compound and fPR (1 .mu.M each) was infused. This
generated a V-V.sub.0 that was approximately twice the value
measured from the mixture. A similar experiment was conducted using
PfR instead, which generated no measurable V-V.sub.0. This confirms
that fPR is indeed the ligand in the mixture. The combination of a
large indicator shift and a smaller than expected V-V.sub.0 for the
ligand is consistent with the presence of additional ligands.
Thrombin is a serine protease capable of cleaving the C-terminal
side of K and R, therefore a large fraction of these peptides serve
as substrates for the enzyme. At the infusion concentrations of the
experiment, these peptides compete with the binding of fPR. This
shows that FC/MS, in conjunction with the indicator analysis, serve
as a rapid means of identifying ligands from mixtures.
[0231] An additional experiment was conducted with this system to
illustrate the use of the roll-up effect in determining the
presence of a strong ligand. A thrombin column similar in construct
to those mentioned above was prepared, this time containing
approximately 50 pmol of active protein. The ligand fPR was
selected as an indicator and infused through the column at a
concentration of 1 .mu.M to generate the selected ion chromatogram
of FIG. 16A. This represents a typical break through curve. The
column was regenerated offline with binding buffer, whereupon a
solution containing 1 .mu.M of fPR and 1 .mu.M of fPR-chloromethyl
ketone (an affinity label with a K.sub.d value of approximately 50
nM) was infused. The selected ion chromatogram is shown in FIG.
16B, where the solid line represents fPR and the dashed line
fPR-chloromethyl ketone. For clarity, the selected ion chromatogram
for the void marker is not displayed. Firstly, there is a time
shift in the break through curve for fPR vs. the break through
curve of FIG. 16A. Secondly, the y axis of FIG. 16B indicates a
maximum intensity of approximately 3 times that of the infusion
concentration, followed by a return to the infusion concentration
level. This indicates the presence of a stronger ligand in the
mixture that causes the release of prebound indicator (this
indicator loaded on to the column ahead of the stronger ligand).
Note that the peak correlates with the onset of the break through
time for the stronger ligand (dashed line). Therefore, monitoring
solely the indicator leads to the identification of a mixture
containing at least on ligand with a binding constant lower than
the indicator.
[0232] From the foregoing description, various modifications and
changes in the composition and method will occur to those skilled
in the art. All such modifications coming within the scope of the
appended claims are intended to be included therein.
* * * * *