U.S. patent application number 15/518174 was filed with the patent office on 2017-11-09 for linkers for protein interaction profiling and methods of making and using the same.
The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF COLORADO, a body corporate. Invention is credited to Christopher BOWMAN, Douglas A. CHAPNICK, Christopher C. EBMEIER, Tao GONG, Tristan D. MCCLURE-BEGLEY, William OLD, Brady WORRELL.
Application Number | 20170321016 15/518174 |
Document ID | / |
Family ID | 55653863 |
Filed Date | 2017-11-09 |
United States Patent
Application |
20170321016 |
Kind Code |
A1 |
OLD; William ; et
al. |
November 9, 2017 |
LINKERS FOR PROTEIN INTERACTION PROFILING AND METHODS OF MAKING AND
USING THE SAME
Abstract
Crosslinking compounds for effective and efficient cross-linking
and identification of intermolecular and intramolecular
interactions of proteins, peptides and nucleic acids.
Inventors: |
OLD; William; (Denver,
CO) ; CHAPNICK; Douglas A.; (Boulder, CO) ;
MCCLURE-BEGLEY; Tristan D.; (Lafayette, CO) ; GONG;
Tao; (Superior, CO) ; WORRELL; Brady;
(Boulder, CO) ; EBMEIER; Christopher C.; (Arvada,
CO) ; BOWMAN; Christopher; (Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF COLORADO, a body
corporate |
Denver |
CO |
US |
|
|
Family ID: |
55653863 |
Appl. No.: |
15/518174 |
Filed: |
October 9, 2015 |
PCT Filed: |
October 9, 2015 |
PCT NO: |
PCT/US15/54965 |
371 Date: |
April 10, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62061973 |
Oct 9, 2014 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C08H 1/00 20130101; C08K
5/10 20130101; G01N 33/6848 20130101 |
International
Class: |
C08H 1/00 20060101
C08H001/00; G01N 33/68 20060101 G01N033/68; C08K 5/10 20060101
C08K005/10 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0001] This invention was made with government support under grant
number CHE1214109, awarded by the National Science Foundation, and
grant number W911NF-14-2-0019, awarded by the U.S. Army Research
Office. The U.S. government has certain rights in the invention.
Claims
1. A crosslinking compound having the chemical structure:
Z-A.sub.n-Z wherein: n is an integer between 1 and 5; A is an atom
or compound; and, each Z is a Michael acceptor, and A is optionally
linked to at least one of an affinity handle (Q), and a molecular
label (L).
2. The crosslinking compound of claim 1, wherein A is phosphorus
(P), sulfur (S), nitrogen (N), oxygen (O), C.sub.1-10 alkyl
optionally substituted with a reactive group or an affinity group,
cycloalkyl, or heteroalkyl or heterocycloalkyl wherein the
heteroatoms are selected from at least one of phosphorus (P),
sulfur (S), nitrogen (N), or oxygen (O).
3-6. (canceled)
7. The crosslinking compound of claim 2, wherein A is pentane or
hexane.
8. The cross-linking compound of claim 1, wherein the Michael
acceptor (Z) is selected from the group consisting of a chemical
moiety conjugated to a protein, peptide or a nucleic acid, an
electron-deficient alkyne, and a chemical moiety that forms a
covalent bond with accessible .epsilon.-amino groups on lysine
residues present in proteins or peptides.
9-12. (canceled)
13. The cross-linking compound of claim 1, comprising at least two
(Z) and wherein at least one Michael acceptor (Z) is capable of
forming a covalent bond with a protein or peptide and at least one
Michael acceptor (Z) is capable of forming a covalent bond with a
nucleic acid.
14. The cross-linking compound of claim 1, comprising at least two
(Z) and wherein each Michael acceptor (Z) is the same chemical
moiety.
15. The cross-linking compound of claim 1, comprising at least two
(Z) and wherein each Michael acceptor (Z) is a unique chemical
moiety.
16. The cross-linking compound of claim 1, wherein each Michael
acceptor (Z) is selected from the group consisting of propiolate,
propiolamide, yn-one, ynethiolate, acrylate, vinylsulfone, and
propargylamide.
17. The cross-linking compound of claim 1, comprising an affinity
handle (Q) that is a chemical moiety that facilitates enrichment of
crosslinker-modified species from a sample selected from the group
consisting of a chemical moiety that facilitates precipitation or
separation of the cross-linking compound from a sample; at least
one of biotin, a histidine residue, and PEG; and, at least one of
an amino acid sequence, polyhistidine, an antibody fragment, and a
nucleic acid sequence.
18-21. (canceled)
22. The cross-linking compound of claim 1, comprising a molecular
label (L) that is at least one chemical entity that renders the
crosslinking compound distinguishable by physical means due to the
presence of the label, selected from the group consisting of
phosphors, radioactive atoms, atomic isotopes, fluorescent dyes,
electron-dense reagents, enzymes, biotin, digoxigenin, haptens or
proteins made detectable by incorporating a metal, radiolabel or
phosphor into the peptide, hydrolases, phosphatases, esterases and
glycosidases, or oxidotases, peroxidases, fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, umbelliferone,
luciferin, 2,3-dihydrophthalazinediones, and a stable isotope.
23. (canceled)
24. (canceled)
25. A cross-linking compound, having the chemical structure
Z-Y-A.sub.n-Y-Z wherein each Z is a Michael acceptor, A is an atom
or compound; n is an integer between 1 and 5; and, each Y is
independently an organic compound of C.sub.1-10 alkyl, optionally
substituted with one or more heteroatoms selected from O, N, S, or
P.
26. The cross-linking compound of claim 25, wherein each linking
group (Y) independently comprises one or more cleavage sites
cleaved by at least one of a chemical cleavage agent, an enzymatic
cleavage agent, or both chemical cleavage agents and enzymatic
cleavage agents.
27. The cross-linking compound of claim 25, wherein each linking
group (Y) comprises an affinity handle (Q), or a molecular label
(L).
28. The cross-linking compound of claim 25, wherein each linking
group (Y) comprises one or more collision-induced dissociation
groups which comprise signature ions formed upon collision-induced
dissociation in mass spectrometric methods.
29. (canceled)
30. The cross-linking compound of claim 28, wherein the
collision-induced dissociation groups comprise fixed-charge
sulfonium ion derivatives of the amino acids methionine and
cysteine, which fragment exclusively via neutral loss of the side
chain CH.sub.3SR, where R is a substituted alkyl group.
31. The cross-linking compound of claim 25 having a molecular
length of between about 1 angstrom and about 50 angstroms.
32. (canceled)
33. (canceled)
34. The cross-linking compound of claim 1, comprising a
dipropiolate.
35. The cross-linking compound of claim 1, comprising at least one
of butane-1,4-diyl dipropiolate; oxybis(ethane-2,1-diyl)
dipropiolate; and di(cylcohexyl-2,1-diyl) dipropriolate.
36-59. (canceled)
60. The crosslinking compound of claim 25, wherein A is phosphorus
(P), sulfur (S), nitrogen (N), oxygen (O), C.sub.1-10 alkyl
optionally substituted with a reactive group or an affinity group,
cycloalkyl, or heteroalkyl or heterocycloalkyl wherein the
heteroatoms are selected from at least one of phosphorus (P),
sulfur (S), nitrogen (N), or oxygen (O).
61. The cross-linking compound of claim 25, wherein the Michael
acceptor (Z) is selected from the group consisting of a chemical
moiety conjugated to a protein, peptide or a nucleic acid, an
electron-deficient alkyne, and a chemical moiety that forms a
covalent bond with accessible .epsilon.-amino groups on lysine
residues present in proteins or peptides.
Description
TECHNICAL FIELD
[0002] This application resides in the field of cross-linkers and
methods of identifying peptide and protein interactions using the
cross-linkers.
BACKGROUND
[0003] Protein interactions represent the foundation of nearly
every aspect of cell signaling. The post-translational modification
of proteins, cytoskeletal rearrangements, and stimulated chemical
communications between cells and tissues are all the direct result
of proteins coming together in tightly defined and orchestrated
geometry resulting in a critical event. Yet, the ability to observe
the consequences of such interactions has radically outpaced the
technology development required to actually capture all the
interactions themselves. For example, the observed changes in
phosphorylation states of proteins are often interrogated with
pharmacological manipulations of classes of protein kinases and
phosphatases, which produce indirect evidence of the relevant
interaction by virtue of its loss coinciding with the measured loss
in the activity of a class of proteins. More information could be
gained, however, if these interactions could be directly trapped
and changes in their frequency and/or geometry observed according
to a relevant state in a cell.
[0004] Popular techniques to monitor protein changes are high
resolution methods such as x-ray crystallography and nuclear
magnetic resonance spectroscopy (NMR). However, these techniques
have limitations. They required large amounts (10-100 mg) of very
pure protein. NMR is also limited by the size of the protein
(.about.40 kD limit), while crystallography requires the formation
of a crystal, which is not always possible and does not mimic
solution behavior, as the crystal is the thermodynamic minima of
the ensemble. Chemical cross-linking is another technique that has
recently been employed to study protein-protein interactions. This
technique has become more prevalent with the coupling of
crosslinking methods with mass spectrometry. These techniques
chemically cross-link proteins through a reaction with a
cross-linker reagent, which creates a stable, covalent linkage
between specific amino acids of the protein. The cross-linker
imposes a distance constraint on the location of protein functional
groups capturing a snapshot of their interaction. This technique
can be used intramolecularly to gain insight into the 3D structure
of a single protein as well. Some of the major advantages to
cross-linking are: 1) it is applicable to low (micromolar)
concentrations of protein; 2) it captures a snapshot of the protein
in its native/dynamic conditions; and 3) it can be easily coupled
to mass spectrometry for detection of cross-linked products and
identification of cross-link site. Chemical cross-linkers have been
widely employed in analysis of three-dimensional protein structures
and protein-protein interactions (PPIs). Formaldehyde and
glutaraldehyde are often employed for their rapid reaction rates
and extensive degree of protein crosslinking, thus, preserving
cells and tissues for histological examinations. The most widely
employed protein crosslinkers use N-hydroxysuccinimide (NHS)
esters, which can undergo rapid S.sub.N2-type reactions with
primary amines. Unfortunately, such activated ester probes are
susceptible to hydrolysis by water, thus compromising their utility
for in vivo crosslinking. Carbodiimide reagents, such as
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC), activate
carboxylic acids and generate new peptide bonds with adjacent amine
residues, resulting in zero-length protein coupling. When
photoactivateable groups, such as diazirines and benzophenones, are
employed as part of heterofunctional protein crosslinkers, reactive
carbenes can easily be liberated which will readily add to any
adjacent residue or abscond hydroxyl groups from water molecules to
form alcohols. Again, such chemical probes, although commonly
employed for the covalent crosslinking of proteins, are highly
susceptible to deleterious non-productive reactions with the
aqueous environment. Recently, technological advances in hybrid
high-resolution mass spectrometry instrumentation and computational
algorithms have allowed for the detection of binding partners and
specific contacting residues with increased sensitivity. For
example, these techniques have been used to explore the regulatory
interactions of a protein phosphatase (PP2A; Herzog et al, Science
337(6100):1348-52; PMID 22984071), the docking geometry of
.beta.-arrestin with .beta.-adrenergic g-protein coupled receptors
(GPCRs) (Shukla et al, Nature 512(7513):218-22; PMID25043026), in
which relatively high-affinity, yet transient interactions between
proteins were successfully captured with chemical crosslinkers.
[0005] With each of the listed crosslinking chemistries (vide
supra), inherent difficulties arise in terms of the conditions used
to maximize the production of informative crosslinks, and the
physiologically relevant states of cells and tissues. For example,
the use of carbodiimides requires proteins to be accessible to a
charged molecule, and cannot, therefore, be employed with much
success in the linking of proteins within selectively permeable
membranes. NHS-esters are prone to spontaneous hydrolysis,
resulting in decreased efficacy and often unpredictable performance
from one experiment to the next. All currently
commercially-available protein crosslinkers designed for use with
mass spectrometry-based protein identification and profiling are
employed to optimal effect at pH values that lay outside those
typically encountered in native cellular compartments, and are
therefore somewhat limited in the nature and diversity of the
interactions they can trap. For this reason, investigation of
protein interactions in vivo typically relies on protein complex
immunoprecipitation ("pull-down" assays) to recover interacting
protein or peptide partners. Alternatively, affinity tags can be
used in genes encoding target proteins to permit efficient
purification from cell lysates. However, many important protein
interactions are weak interactions that are not strong enough to be
identified by these methods. Thus, there is a strong desire for
chemical cross-linking compounds and methods of using the
compounds, particularly in vivo, to freeze protein interactions by
forming covalent bonds, allowing sample analysis, optionally
combined with other protein purification techniques in an aqueous
environment.
SUMMARY
[0006] The present disclosure describes the conception, synthesis,
and application of a novel protein crosslinking chemistry, based on
the use of electron-deficient alkyne/propiolate Michael acceptors
which react selectively with amine- and sulfhydryl-bearing amino
acids. The cross-linker compounds are useful in methods for
preparing one or more cross-linked biomolecules, biomolecular
complexes of two or more biomolecules, and fragments from such
cross-linked biomolecules and/or biomolecular complexes, as well as
methods for identifying cross-links in such cross-linked
biomolecules and/or biomolecular complexes. One aspect of the
disclosure is a crosslinker compound having the chemical
structure:
Z-(Y)-A.sub.n-(Y)-Z
wherein: n=1-5; A is an organic compound that may be optionally
substituted with one or more collision-induced dissociation groups,
and one or more affinity handles. Each A can act independently or
as a Q or L, wherein Q is an affinity handle and L is a molecular
label. Each A may be joined to Z through one or more optional
linking group Y, wherein each Y is an organic compound of
C.sub.1-10, optionally substituted with one or more heteroatoms
selected from O, N, S, or P and one or more collision-induced
dissociation groups, and one or more affinity handles.
[0007] The Michael acceptor (Z) of these compounds may include at
least one compound selected from propiolate,
propiolamide/propargylamide, ynone, ynethiolate, acrylate, and
vinylsulfone. In certain embodiments, Z is at least one of:
##STR00001##
[0008] The affinity handles (Q) may include at least one compound
selected from biotin, streptavidin, PEG, antibody, organic azide,
or non-Michael accepting alkyne. The molecular labels (L) may
include at least one compound selected from phosphors, radioactive
atoms, fluorescent dyes, electron-dense reagents, enzymes, biotin,
digoxigenin, haptens or proteins made detectable by incorporating a
metal, radiolabel or phosphor into the peptide, hydrolases,
phosphatases, esterases and glycosidases, or oxidotases,
peroxidases, fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, umbelliferone, luciferin, and
2,3-dihydrophthalazinediones,
[0009] The linking group (Y) may be chosen to have a molecular
length of between 1 angstrom and 50 angstroms, preferably between
about 5 angstroms and about 20 angstroms, most preferably about 12
angstroms.
[0010] In some embodiments, the crosslinking compound is a
dipropiolate. In certain embodiments, the crosslinking compound is
at least one of butane-1,4-diyldipropiolate;
oxybis(ethane-2,1-diyl) dipropiolate; and di(cylcohexyl-2,1-diyl)
dipropriolate.
##STR00002##
Specific Examples
##STR00003##
[0012] Another aspect of the invention provides methods of
crosslinking at least one protein or peptide, which include mixing
a sample containing the at least one protein or peptide with a
crosslinker compound of this disclosure to form a crosslinked
protein or peptide; fragmenting the crosslinked protein or peptide
to form fragmented peptides; introducing the fragmented peptides
into a mass spectrometer and detecting a mass-to-charge ratio of
the fragmented peptides.
[0013] In these methods, the mixing step may be conducted at pH
between pH 5 and pH 10, preferably between pH 6 to pH 9, and more
preferably between pH 6 to pH 8. In certain embodiments, the mixing
step may be conducted at a physiological pH of about 7.4. In these
methods, the fragmenting step may include digesting the
cross-linked sample using a protease.
[0014] These methods may further comprise using the detected
mass-to-charge ratio of the fragmented peptides to determine an
amino acid sequence associated with the fragmented peptides.
[0015] These methods may further comprise using the detected
mass-to-charge ratio of the fragmented peptides to identify
intermolecular and intramolecular protein interactions in a sample
comprising the at least one protein or peptide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1a is a coomassie stained polyacrylamide gel showing
evidence of crosslinked yeast ADH and characteristic shifts in
electrophoretic protein migration.
[0017] FIG. 1b is a graphical representation of crosslinker
performance quantitated from coomassie stained band densities
comparing disuccinimidyl suberate with butyldipropriolate.
[0018] FIG. 2a is a coomassie stained polyacrylamide gel showing
changes in yeast ADH protein electrophoretic mobility following
crosslinking with butyldipropriolate or disuccinimidyl suberate at
various pH.
[0019] FIG. 2b is a graphical representation of crosslinker
performance at different pH quantitated by coomassie band
density.
[0020] FIG. 3 is a coomassie stained polyacrylamide gel showing the
tendency of disuccinimidyl suberate to generate nonspecific protein
crosslinks between bovine serum albumin and yeast ADH, and the
relative lack of nonspecific crosslinks produced by
butyldipropriolate.
[0021] Tables 1a and 1b are tabulations of protein:protein
crosslinks identified by LC-MS/MS analysis of proteins present in
gel bands excised from the polyacrylamide gel pictured in FIG.
3.
[0022] FIG. 4 is a coomassie stained polyacrylamide gel showing
characteristic electrophoretic mobility shifts of yeast ADH
following crosslinking by butyldipropriolate,
oxybis(ethane-2,1-diyl) dipropiolate, and di(cylcohexyl-2,1-diyl)
dipropriolate.
[0023] FIG. 5 is an IUPRED depiction of intrinsic protein disorder
in the M3-M4 subunit of the mouse .alpha.4 nicotinic acetylcholine
receptor subunit.
[0024] Tables 2a and 2b are tablulations of inter-subunit
crosslinks identified for .alpha.4.beta.2 nicotinic receptors that
map to the extracellular and intracellular domains under control
cell culture conditions and following 24 hour incubation with 1
.mu.M nicotine to induce desensitized receptor conformations.
DETAILED DESCRIPTION
[0025] Aspects of the present invention provide crosslinking
compounds having electron-deficient alkyne protein-reactive groups,
which crosslink proteins/peptides through direct coupling of amino
groups on lysine residues, similar to NHS-ester based crosslinkers,
but with superior performance across a physiologically relevant
range of pH and with substantially reduced nonspecific protein
conjugation. These crosslinking compounds are useful for the
investigation of protein-protein interactions (PPIs),
three-dimensional protein structures, as well as protein-nucleic
acid (DNA or RNA) interactions, in addition to being useful as
protein conjugation reagents. Analysis of these structures and
interactions can lead to identification of ligand binding sites on
proteins and associated conformational sites that provide insight
into many applications, such as molecular docking, de novo drug
design and structural identification and comparison of functional
sites.
[0026] These crosslinking compounds afford distinct advantages over
other currently available cross-linkers. For example, the inventors
have demonstrated that Michael acceptor protein-reactive groups are
less dependent on alkaline pH than amine-specific crosslinking
reagents based on N-hydroxysuccinimide ester (NHS ester)
crosslinkers (such as disuccinimidyl suberate; DSS) and, therefore,
are more likely to preserve relevant protein:protein interactions
across cellular compartments and organelle environments that may be
selected against profiling with other commercially available
reagents.
Crosslinking Compounds
[0027] The crosslinking compounds of this disclosure include
compounds having the chemical structure:
Z-(Y)-A.sub.n-(Y)-Z
wherein n is an integer between 1 and 5. A is an atom or compound
which serves as a central atom to which additional reactive
group(s) and/or affinity group(s), as defined below, can be
attached. In certain embodiments, A is phosphorus (P), sulfur (S),
nitrogen (N), or oxygen (O).
[0028] Each Y is an optional C.sub.1-10 alkyl or aromatic spacer to
attach reactive group(s) and/or affinity group(s), as defined
below. Such substituted C.sub.1-10 alkyl may include cycloalkyl,
such as pentane or hexane, as well as heteroalkyl and
heterocycloalkyl, including common organic heteroatoms, such as
phosphorus (P), sulfur (S), nitrogen (N), or oxygen (O). Aromatic
spacers, including benzene, extended aromatic systems, or
heterocycles including common organic heteroatoms as listed above
are also covered by this claim.
[0029] Each A can act independently or as a Q or L, when Q is an
affinity handle and L is a molecular label.
[0030] The Michael acceptor (Z) of these compounds is a chemical
moiety that is capable of conjugating to a protein, peptide and/or
a nucleic acid. Z is an electrophile that forms a covalent bond
with a donor moiety (i.e., a nucleophile) in the target compound
via a Michael reaction. In certain embodiments, the Michael
acceptor electrophile (Z) is an electron-deficient alkyne.
[0031] Thus, the donor moiety present in the target compound is a
nucleophile that reacts with the Michael acceptor (Z) via a Michael
reaction to form a covalent bond between the crosslinking compound
and the target compound. Examples of typical target compounds
include proteins, peptides or nucleic acids (DNA or RNA)
(collectively "biomolecules") that are of interest for crosslinking
via the methods of this disclosure). The Michael acceptor (Z) often
reacts with lysine and free cysteine moieties present in proteins
or peptides, for example accessible .epsilon.-amino groups on
lysine residues.
[0032] As multiple, independent moieties (Z) may be present in the
crosslinking compounds of this disclosure, these crosslinking
compounds are capable of simultaneously conjugating to a protein or
peptide and a nucleic acid. For example, Michael acceptors (Z) that
are capable of conjugating to proteins or peptides react with a
specific amino acid of the peptide or protein. Similarly, Michael
acceptors (Z) that are capable of conjugating to a nucleic acid
molecule (such as DNA or RNA) react with at least one specific
nucleic acid base or other chemical functionality in the nucleic
acid molecule.
[0033] In some embodiments, each Michael acceptor (Z) is/are the
same, thereby rendering the cross-linker compound homobifunctional,
homotrifunctional, etc. That is, when each Michael acceptor (Z) is
the same, the crosslinker makes two of the same conjugations to the
peptide(s), protein(s) or nucleic acid molecules to be
cross-linked.
[0034] In other embodiments, each Michael acceptor (Z) is/are
different, i.e., the crosslinker is heterobifunctional,
heterotrifunctional, etc., and makes two different conjugations to
the peptides(s), protein(s) or nucleic acid molecules to be
cross-linked.
[0035] In some embodiments, when each Michael acceptor (Z) is/are
different, at least one Z may be an electrophilic Michael acceptor
group that is capable of conjugating to an amino acid of a protein
or peptide, and another Z is capable of conjugating to a nucleic
acid base, or other chemical functionality of a nucleic acid
molecule. In this way, a heterobifunctional crosslinker having a
Michael acceptor (Z) capable of conjugating to an amino acid (e.g.
an amine-reactive group) and a Michael acceptor (Z) capable of
conjugating to a nucleic acid base (e.g. a thymine-reactive group),
is capable of conjugating to both a protein or peptide and a
nucleic acid molecule.
[0036] Preferred embodiments of crosslinking compounds of this
disclosure include a Michael acceptor (Z) that include at least one
compound selected from propiolate, propiolamide/propargylammide,
ynone, ynethiolate, acrylate, and vinylsulfone. These chemical
moieties are readily understood by those of skill in the art
familiar with organic chemistry and Michael reactions and are
readily available as starting materials for synthesis of these
crosslinking molecules. Other Michael acceptor (Z) groups are
available that would be suitable for alternative embodiments of the
subject matter of this disclosure. Those of skill in the art will
understand that any suitable Michael acceptor, now known or
hereafter developed, may be used in forming the crosslinking
compounds described herein.
[0037] Affinity handle (Q) refers to a chemical moiety that may be
attached to the crosslinker compounds of this disclosure to
facilitate enrichment of crosslinker-modified species from a sample
that may include mixtures of unmodified compounds (i.e.,
non-crosslinked compounds) as well as other biological or
non-biological molecules present in a sample containing
biomolecules of interest. The affinity handle(s) enrich the
cross-linked molecule(s) by precipitation or separation of the
"handle" moiety. For example, an affinity handle may be
precipitated by its corresponding binding moiety (e.g., biotin is
precipitated by avidin; histidine is precipitated by nickel, and an
antibody is precipitated by its antigen). In related embodiments,
the affinity handle (Q) may be absent from the cross-linking
compounds of this disclosure but a target protein may naturally
express, or be engineered to express, an affinity handle and the
crosslinked protein may be enriched using that affinity handle.
[0038] These affinity handles are readily understood by those of
skill in the art familiar with organic chemistry and protein and
peptide purification methods and are available as starting
materials or in kits from various manufacturers for synthesis of
these crosslinking molecules. Examples may include, but are not
limited to, amino acid sequences (e.g., polyhistidine or antibody
fragments), small-molecules (e.g. biotin), or nucleic acid
sequences (e.g., DNA or RNA). Exemplary affinity handles include at
least one compound selected from biotin, streptavidin, PEG,
antibodies, or similar tags. Those of skill in the art will
understand suitable affinity groups that can be used to further
isolate or purify the crosslinked compounds formed in a sample or
composition.
[0039] Molecular labels (L) are one or more chemical entities that
render the crosslinking compounds of this disclosure (and therefore
crosslinked compounds in a sample or composition) distinguishable
by some physical means (e.g. by mass) because of the presence of
the label. Non-limiting examples of molecular labels (L) may
include at least one compound selected from phosphors, radioactive
atoms, atomic isotopes, fluorescent dyes, electron-dense reagents,
enzymes, biotin, digoxigenin, haptens or proteins made detectable
by incorporating a metal, radiolabel or phosphor into the peptide,
hydrolases, phosphatases, esterases and glycosidases, or
oxidotases, peroxidases, fluorescein and its derivatives, rhodamine
and its derivatives, dansyl, umbelliferone, luciferin, and
2,3-dihydrophthalazinediones. Exemplary isotopes that may be used
as the molecular label (L) include, without limitation: carbon
(C.sup.12 and C.sup.13), nitrogen (e.g., N.sup.14 and N.sup.15),
sulfur (e.g., S.sup.32 and S.sup.34), oxygen (e.g., O.sup.16,
O.sup.17 and O.sup.18), bromine (Br.sup.79 and Br.sup.81), or
chloride (e.g., Cl.sup.35 and Cl.sup.37).
[0040] Each A may be joined to Z through one or more optional
linking group(s) (Y), wherein each Y is independently an organic
compound of C.sub.1-10, optionally substituted with one or more
heteroatoms selected from O, N, S, or P. Each Y may independently
include a cleavable linker portion that may include one or more
cleavage sites, which may be cleaved by chemical cleavage agents,
enzymatic cleavage agents, or both chemical cleavage agents and
enzymatic cleavage agents. When present in the crosslinking
compounds of this disclosure, the linking group(s) (Y), may be
substituted or appended to include the functional affinity handles
(Q) or molecular labels (L) in addition to one or more cleavage
sites.
[0041] Additionally, these linking group(s) (Y) may include one or
more collision-induced dissociation groups. Collision-induced
dissociation groups may include signature ions (e.g., the
precursors of signature ions) formed upon CID, particularly in
CID/MS/MS methods. The presence of such CID groups may provide
greater specificity in protein or peptide identification in the
methods of this disclosure, compared to that of pure MS-based
methods. Examples of such CID dissociation groups include
fixed-charge sulfonium ion derivatives of the amino acids
methionine and cysteine, which fragment exclusively via neutral
loss of the side chain CH.sub.3SR, where R is a substituted alkyl
group. The optional linking group (Y) may be chosen to have a
molecular length of between about 1 angstrom and about 50
angstroms, preferably between about 5 angstroms and about 20
angstroms, more preferably about 12 angstroms.
[0042] In some embodiments, the crosslinking compound of this
disclosure is a dipropiolate. In certain embodiments, the
crosslinking compound is at least one of butane-1,4-diyl
dipropiolate; oxybis(ethane-2,1-diyl) dipropiolate; and
di(cylcohexyl-2,1-diyl) dipropriolate.
Methods of Cross-Linking and Analysis
[0043] Using the disclosed methods, a protein or a protein complex
may be analyzed to elucidate protein structure or the interaction
between proteins or protein complexes, when used in combination
with mass spectrometry.
[0044] Various methods may be used to investigate the crosslinked
biomolecules to evaluate the protein or nucleic acid molecules and
their interactions. Essentially, crosslinkers are added into the
interacting proteins and/or nucleic acids. The crosslinkers capture
the interactions by forming covalent chemical bonds with accessible
amino acid residue side chains on the biomolecules. The crosslinked
biomolecules are enzymatically digested directly in solution or in
gel bands after separation. By analyzing the digest on a mass
spectrometer (that may include LC separation), the crosslinked
molecules can be detected because they have modified mass due to
the crosslinker. With MS/MS spectra and pre-knowledge of which
amino acid side chains the crosslinker reacts with, the exact amino
acid residues that are crosslinked are determined. This analysis
step may be more efficiently completed with computer algorithms
than with manual processing because of the numerous possibilities
for crosslinking peptides and nucleic acids. Both commercial and
customized computer programs are available for this purpose.
[0045] This analysis (based on the MS data) determines which
residues in the biomolecules are crosslinked to each other. In the
protein complex structure, these residues should be apart from each
other at the distance of the length of the crosslinker used in the
study. This spatial constraint information can be combined with
mutation studies and structures from X-ray crystallography or NMR
to further examine aspects of the binding interface. Additionally,
computational docking structure data may provide additional insight
into the interactions of the crosslinked biomolecules and/or
confirm the results of this crosslinking-MS analysis.
[0046] Thus, in some embodiments, methods of crosslinking a
biomolecule of this disclosure includes mixing a sample or
composition having at least one protein, peptide or nucleic acid of
interest with a crosslinker compound of this disclosure. The mixing
may be conducted in vivo, in vitro, or in situ. A variety of
samples may be analyzed for protein interactions. The samples are
obtained from cell lysate or cell culture or a clinically relevant
tissue or serum sample. In certain embodiments, one or more
proteins are cross-linked inside substantially intact cells before
isolation and subsequent analysis. Suitable conditions for the
crosslinking in the mixing step may include a pH in the
physiological range of the protein, peptide or nucleic acid
molecules of interest. Thus, the mixing step may be conducted at pH
between pH 5 and pH 10, or between pH 6 to pH 9, or between pH 6 to
pH 8. In certain embodiments, the mixing step may be conducted at a
pH of about 7.4.
[0047] After mixing in order to generate a sample containing
crosslinked molecules for MS analysis, the sample may be further
processed or purified (for example gel electrophoresis, and
subsequent enzymatic digestion/reduction of a crosslinked
product),
[0048] In certain embodiments, the cross-linked sample is digested
with an enzymatic or chemical cleaving agent(s). This digestion may
include the step of fragmenting the cross-linked biomolecules,
biomolecular complexes or mixtures thereof. The fragments are
digested to obtain cross-linked species from the cross-linked
biomolecules or biomolecular complexes of sufficient small size for
further analysis. Cross-linked fragments can be prepared by any
method known in the art for cleaving large biomolecules into
smaller fragments. Such fragmentation can be performed by chemical
protein cleavage or treatment of proteins with proteolytic enzymes
as to obtain smaller size fragments. Fragmentation using selective
cleavage reagents results in fragments that are more easily
identified than fragments obtained by a non-selective cleavage.
Examples of specific chemical cleavage agents for proteins and
peptides are CNBr, which cleaves proteins at methionine residues,
and dilute acid at pH 2, which cleaves specifically at aspartate
residues at high temperature, e.g. 108.degree. C. Examples of
specific proteases are trypsin, cleaving at lysine and arginine
residues and Glu-C endoproteinase, cleaving at glutamate and, to a
lesser extent, at aspartate residues. Efficient cleavage of
cross-linked protein complexes can also be performed with pepsin,
cleaving preferentially in hydrophobic segments with poor residue
selectivity. In complex structures, identification of peptic
peptides is more time-consuming than identification of peptides
obtained by selective cleavage. Fragmentation can also be achieved
by using a combination of two or more of such cleavage methods. For
sequence specific cleavage of DNA in protein-DNA cross-links,
restriction enzymes can be employed. However, for cross-linked
protein-DNA or protein-RNA complexes, non-specific nucleases, such
as DNase I and the RNase .alpha.-sarcin, can be employed to
decrease the size of the cross-linked nucleic acid moiety.
[0049] In order to purify or isolate the crosslinked biomolecule,
the crosslinked complexes may be separated by, for example, liquid
chromatography or gel electrophoresis. Additionally or
alternatively, the crosslinked complexes including an affinity
handle as part of the crosslinking compound may be precipitated,
captured, or separated by capturing the affinity handle, thereby
capturing the crosslinked complex or complex fragments. For
example, a biotin affinity handle may be captured or enriched by
avidin affinity chromatography. As would be understood by those of
ordinary skill in the art, the complexity of the sample will
determine the need to perform one or more of these additional
purification steps. Also, when needed, those of ordinary skill in
the art would be able to determine which purification steps to
employ and how to employ them, based on the identity of a specific
affinity handle within the crosslinking compound employed.
[0050] The crosslinked biomolecules, or fragments thereof, may be
dissociated to form a dissociated sample. The dissociation process
may be any suitable dissociation method including, but not limited
to, collisions with an inert gas (known as collision-induced
dissociation (CID or collisionally-activated dissociation (CAD);
(ii) collisions with a surface (known as surface-induced
dissociation or SID); (iii) interaction with photons (e.g. via a
laser) resulting in photodissociation; (iv) thermal/black body
infrared radiative dissociation (BIRD), and (v) interaction with an
electron beam, resulting in electron-induced dissociation for
singly charged cations (EID), electron-capture dissociation (ECD)
for multiply charged cations, or combinations thereof.
[0051] The crosslinked biomolecules, or fragments thereof, may also
be ionized by methods that may include, but are not limited to,
electrospray ionization (ESI), matrix-assisted laser desorption
ionization (MALDI), or fast atom bombardment (FAB). The ionized
sample may subsequently be activated to fragment the ions of the
ionized sample. In some embodiments, this activation
(fragmentation) is carried out by collision induced dissociation
(CID), electron transfer dissociation (ETD), pulsed Q dissociation
(PQD), high energy C-trap dissociation (HCD) or CID-HCD. Tandem
mass spectrometry (MS/MS) instrumentation allows for the
implementation of ionization and fragmentation. In certain
embodiments, the crosslinked biomolecules, or fragments are passed
through a first mass resolving spectrometer to select precursor
protein or peptide ions having a first desired mass-to-charge
ratio; subjecting the precursor ions of the first mass to charge
ratio to dissociation to form a product ion having a second
mass-to-charge ratio and detecting the product ions.
[0052] Analysis of the mass-to-charge ratio results can efficiently
and accurately determine the intramolecular and intermolecular
(protein, peptide and/or nucleic acid) interactions associated with
a crosslinked biomolecule. The methods of analysis of the present
disclosure may be used for amino acid, peptide or protein
identification, differential quantitation, analysis of post
translational modification status, and analysis of cross-linking
status or interaction of proteins.
Synthesis of Crosslinker Compounds
[0053] The synthesis of a crosslinker compound of this disclosure
is carried out using known methods of organic synthesis, as
described in the Examples and references cited herein. For example,
The Molecular Probes Handbook, 11th Edition, Cross-linking and
Photoactivatable Reagents, Chapter 5, Section 5.1 Invitrogen Life
Science; and Bioconjugate Reagents, Bioconjugate Techniques, 2nd
Edition, by Greg T. Hermanson, Published by Academic Press, Inc.,
2008; Rostovtsev et al., Angew. Chem.-Int. Edit. 2002, 41, 2596;
Tornoe et al., J. Org. Chem. 2002, 67, 3057; Baskin et al., Proc.
Natl. Acad. Sci. U.S.A. 2007, 104, 16793; Jewett and Bertozzi,
Chem. Soc. Rev., 2010, 39, 1272, Szychowski et al., J. Am. Chem.
Soc. 2010, 132, 18351 and Brittain et al., Nat. Biotechnol. 2005,
23, 463, all of which are incorporated herein. Chemists of ordinary
skill in the art can modify the synthesis scheme based on the
selection of variable groups in the crosslinking compounds of this
disclosure.
[0054] The following Examples are presented for illustrative
purposes only, and do not limit the scope or content of the present
application.
EXAMPLES
Example 1 In Vitro Crosslinking
[0055] To study whether electron-deficient alkyne Michael acceptors
act as efficient amine-reactive protein crosslinkers, the inventors
used a highly purified homotetrameric protein complex (ADH) as a
test system for the utility of novel Michael acceptors in
stabilizing protein:protein interactions via covalent coupling of
accessible .epsilon.-amino groups on proximal lysine residues. ADH
exists under non-denaturing conditions as a stable homotetramer,
but following solubilization in chaotropic buffers (such as SDS in
excess of 1%), the complex dissociates into linear monomers. ADH
complexes incubated with 1 mM butyldipropiolate, then quenched with
10 mM ethanolamine showed patterns of migration with 10% SDS-PAGE
indicative of molecular weight shifts consistent with the covalent
coupling of dimeric proteins (FIG. 1a, black boxes). Varying the
concentration of crosslinker within the preparation and keeping the
DMSO % constant showed a highly concentration-dependent effect of
crosslinker performance, with minimal protein crosslinking present
at concentrations below 100 .mu.M (FIG. 1b). The performance of
butyldipropiolate was comparable to that of the commercial standard
NHS-ester protein crosslinker DSS in terms of 1/2-maximal
concentration. ADH should never exist under native conditions as
anything more massive than a tetramer of 146 kDa in total mass,
however, DSS produces a very apparent "ladder" of crosslinked ADH
(FIG. 1; arrows), indicating substantial protein polymerization
that reflects relatively high nonspecific crosslinking of proteins
in solution. Gel bands were excised to isolate the apparent protein
dimers from both DSS-crosslinked and butyldipropiolate-crosslinked
gels, and used for in-gel digestion and mass spectrometry to
sequence the crosslinked peptides and assign some distance
parameters based on the identification of crosslinks between the
peptide chains. Interrogation of the peptide chains observed to be
crosslinked by butyldipropiolate indicates the crosslinks
identified by StavroX can be mapped onto existing crystal
structures, which shows that butyldipropiolate produces reliable
crosslinks between adjacent peptide chains in ADH. This example
confirms that effective protein crosslinks are created through
direct coupling of .epsilon.-amino groups on proximal lysine
residues, similar to NHS-ester based crosslinkers like DSS.
Example 2 pH Sensitivity
[0056] NHS-ester protein reactive groups have been shown to be
highly dependent on reaction buffer pH, and most commercial
preparations suggest pH<8.0 for efficient reactions with
.epsilon.-amines of lysine, since the deprotonated form is more
likely to participate in the SN2 reaction with NHS. Because the use
of Michael acceptor reactive groups may be less sensitive to
variations in pH within a physiological range (6-8), the inventors
used PBS at pH 6.5, 7.5, 8.5, 9.5 to probe the performance of DSS
versus butyldipropiolate in crosslinking ADH across a 4-log pH
range. The inventors observed ADH was crosslinked with equivalent
efficiency at pH 6.5, 7.5, and 8.5, with no apparent protein
observed on the gel at pH<9, indicating a nonspecific effect of
very high pH on crosslinking efficiency for both DSS and
butyldipropriolate (FIG. 2a, black boxes). DSS, by contrast, showed
very high sensitivity to pH, with a linear decline in crosslinking
efficiency observed from pH 8.5 to pH 6.5 (FIG. 2b).
[0057] When the crosslinker efficiency is substantially reduced in
acidic pH relative to alkaline, as is the case with known NHS-ester
linking compounds, identifying protein:protein interactions will be
systematically biased towards some compartments and eliminated for
others, drastically limiting the physiological relevance of any
large-scale interactome analysis that depends on stabilizing
transient interactions. This example shows that the Michael
acceptor crosslinking compounds of this disclosure are less
dependent on alkaline pH than NHS-ester crosslinkers (like DSS) and
therefore are more likely to preserve relevant protein:protein
interactions across cellular compartments and organelle
environments that may be selected against profiling with other
commercially available reagents.
Example 3 Crosslinking Specificity
[0058] Because the inventors observed very little protein
"laddering" with butyldipropiolate crosslinking of ADH in solution
compared to equivalent concentrations of DSS, the inventors decided
to evaluate the nonspecific crosslinking between the two compounds
using equimolar mixtures of ADH (protein tetramer) with BSA
(protein monomer) and crosslinking under the conditions established
with previous experiments. After crosslinking with either DSS or
butyldipropiolate at 1 mM in PBS pH 7.4, 10% DMSO, SDS-PAGE showed
very obvious nonspecific crosslinking of BSA and ADH with DSS in
dose-dependent fashion (FIG. 3, insert box "a") and very little
with butyldipropiolate (FIG. 3 insert box "b"). Analysis of
crosslinked peptides with LC-MS/MS from excised gel bands
(indicated by arrows in FIG. 3), shows that with ADH and BSA
mixtures crosslinked with DSS, 9 out of 10 top-scoring crosslinked
peptides reflect nonspecific crosslinking, either linking BSA to
BSA, or ADH to BSA. With ADH and BSA mixtures crosslinked with
butyldipropiolate, only 3 out of 10 top-scoring crosslinked
peptides reflected nonspecific reactions (Table 1a) as opposed to
DSS, where 9 out of 10 identified protein crosslinks indicated
nonspecific reactivity between non-meaningful interactions (Table
1b).
TABLE-US-00001 TABLE 1a Identified DSS Crosslinks Score m/z. z Mass
meas. Mass calc. Dev (pp . . . Peptide (1) Protein (1) 99 778.414
+2 1555.82 1555.816 2.88 [GKY] >sp|P02769|ALB . . . 86 581.627
+3 1742.866 1742.864 1.57 [GKYLY] >sp|P02769|ALB . . . 86
581.627 +3 1742.866 1742.864 1.57 [GKYLY] >sp|P02769|ALB . . .
48 655.997 +3 1995.977 1995.981 -2.12 (mKWVTF] >sp|P02769|ALB .
. . 44 633.986 +3 1899.943 1899.941 1.31 [SQY] >sp|P02769|ALB .
. . 43 778.414 +2 1555.82 1555.816 2.88 [WGKY] >sp|P02769|ALB .
. . 42 633.986 +3 1899.943 1899.941 1.31 [SQY] >sp|P02769|ALB .
. . 25 633.986 +3 1899.942 1899.941 0.64 [SQY] >sp|P02769|ALB .
. . 25 633.986 +3 1899.942 1899.941 0.64 [SQY] >sp|P02769|ALB .
. . 21 851.715 +3 2553.13 2553.128 0.89 [AKAmGY] >sp|P00330|AD .
. . Score From ( . . . To (1) Peptide (2) Protein (2) From ( . . .
To (2) 99 159 161 [KADEKK . . . >sp|P02769|ALB . . . 151 158 86
159 163 [ESHGKL . . . >sp|P00330|AD . . . 14 21 86 159 163
[ESHGKL . . . >sp|P00330|AD . . . 14 21 48 0 6 [YAPELLYY
>sp|P02769|ALB . . . 173 180 44 52 54 (mSIPET . . .
>sp|P00330|AD . . . 0 12 43 158 161 [KADEKKF] >sp|P02769|ALB
. . . 151 157 42 52 54 (mSIPET . . . >sp|P00330|AD . . . 0 12 25
52 54 (mSIPET . . . >sp|P00330|AD . . . 0 12 25 52 54 (mSIPET .
. . >sp|P00330|AD . . . 0 12 21 191 196 [AGIKWL . . .
>sp|P00330|AD . . . 89 103
TABLE-US-00002 TABLE 1b Identified butyldipropriolate Crosslinks
Score m/z. z Mass meas. Mass calc. Dev (pp . . . Peptide (1)
Protein (1) 29 711.684 +3 2133.038 2133.043 -2.33 [KIGDY]
>sp|P00330|AD . . . 29 835.745 +3 2505.222 2505.222 -0.09
[ESHGKL . . . >sp|P00330|AD . . . 29 835.745 +3 2505.222
2505.222 -0.09 [ESHGKL . . . >sp|P00330|AD . . . 29 835.745 +3
2505.222 2505.222 -0.09 [ESHGKL . . . >sp|P00330|AD . . . 21
922.708 +4 3687.811 3687.811 -0.09 [ANKYNG . . . >sp|P02769|ALB
. . . 20 627.061 +4 2505.221 2505.222 -0.47 [ESHGKL . . .
>sp|P00330|AD . . . 20 627.061 +4 2505.221 2505.222 -0.47
[ESHGKL . . . >sp|P00330|AD . . . 20 627.061 +4 2505.221
2505.222 -0.47 [ESHGKL . . . >sp|P00330|AD . . . 20 627.061 +4
2505.221 2505.222 -0.47 [ESHGKL . . . >sp|P00330|AD . . . 18
1155.31 +4 4618.218 4618.223 -1.11 [JDLGEE . . . >sp|P02769|ALB
. . . Score From ( . . . To (1) Peptide (2) Protein (2) 29 84 88
[APELLYY . . . >sp|P02769|ALB . . . 29 14 21 (MSIPET . . .
>sp|P00330|AD . . . 29 14 21 (MSIPET . . . >sp|P00330|AD . .
. 29 14 21 (MSIPET . . . >sp|P00330|AD . . . 21 181 188 [DKLKHL
. . . >sp|P02769|ALB . . . 20 14 21 (MSIPET . . .
>sp|P00330|AD . . . 20 14 21 (MSIPET . . . >sp|P00330|AD . .
. 20 14 21 (MSIPET . . . >sp|P00330|AD . . . 20 14 21 (MSIPET .
. . >sp|P00330|AD . . . 18 36 51 [KIGDYAG.. >sp|P00330|AD . .
.
[0059] These results point to a unique property of protein
crosslinking via rapid Michael addition using electron-deficient
alkynes as Michael acceptor groups for protein conjugation, and
this example demonstrates the use of electron-deficient alkyne
crosslinking compounds of this disclosure similar to NHS-ester
based crosslinkers, but with substantially reduced nonspecific
protein conjugation.
Example 4 Analysis of Heteromeric Protein Interactions
[0060] The inventor next investigated whether the superior
performance of butyldipropiolate could be used to explore sites of
interaction between purified heteromeric protein complexes. The
inventors employed a purified Skp1-Skp2 (Uniprot P63208/Q13309),
which forms part of the SCF E3-ubiquitin ligase complex. This
complex represented a particularly salient application of protein
crosslinking and mass spectrometry-based interaction
identification, as Skp1 possesses a disordered C-terminal region
that is not part of any current high-resolution structural
profiles. The inventors were able to successfully identify two
sites of interaction between proximal lysine residues on Skp1 and
Skp2, including a crosslink between the C-terminal lysine of Skp1
that appears to participate in a relevant interaction geometry with
Skp2 that has not been previously described.
[0061] Profiling the structural determinants of these functionally
relevant interactions with "gold standard" techniques such as
electron microscopy and NMR is difficult when the target protein
contains intrinsically disordered regions, such as in this
Skp1-Skp2 model previously studied. The C-terminus of the S-phase
kinase associated protein (Skp1) contains such a disordered region,
and with crosslinker compounds of this disclosure, the inventors
were able to demonstrate that a disordered region previously
inaccessible to other techniques was contributing to a stable
interaction with its requisite partner, Skp2. Thus, this example
demonstrates the superior utility of the crosslinker compounds of
this disclosure in elucidating intramolecular and intermolecular
protein interactions.
Example 5 Crosslinker Variations
[0062] The previous experiments established the utility of the
novel electron-deficient alkyne reactive groups of the crosslinking
compounds of this disclosure to participate in Michael addition to
free amines on lysine residues of purified proteins. The inventors
next sought to explore the effects of alterations to the structural
rigidity and length of the alkyl chain connecting the two reactive
groups. Expanding the length of the chain and decreasing the
hydrophobicity by replacing the 1-4-butanediol with an expanded
polyethylene glycol (PEG), resulted in oxytris(ethane-2,1 diyl)
dipropriolate. Increasing structural rigidity and maintaining
hydrophobicity by using a dicyclohexane linker produced
di(cylcohexyl-2,1-diyl) dipropriolate. In vitro crosslinking of ADH
with butyldipropiolate, oxytris(ethane-2,1 diyl) dipropriolate, and
di(cylcohexyl-2,1-diyl) dipropriolate, with subsequent analysis of
apparent migration shifts with SDS-PAGE (FIG. 4) indicated that
concentration-dependent crosslinking performance was slightly
reduced for oxytris(ethane-2,1 diyl) dipropriolate, and more
severely attenuated for di(cylcohexyl-2,1-diyl) dipropriolate in
comparison to the prototype molecule (butyldipropiolate).
Example 6 In Vivo Crosslinking
[0063] An important application for protein crosslinker reagents is
their use in the preservation and subsequent profiling of protein
complexes participating in dynamic cell signaling events within
living cells. The inventors used a derivative of B35 rat
neuroblastoma cell line (LTR1-1d) that stably expresses
.alpha.4.beta.2 nAChRs (the major neuronal subtype) to crosslink
these low-abundance protein complexes in their native environment.
The .alpha.4.beta.2 nAChR is a heteropentameric ligand-gated ion
channel complex, which is assembled in alternate stoichiometries
(.alpha.4(2).beta.2(3) and .alpha.4(3).beta.2(2)) to form receptors
of high- and low-sensitivity to agonist activation, respectively
(Zhou et al, J Neurosci 23(27):9004-15; PMID 14534234; others).
Functional parameters of nAChR activation and desensitization have
been interrogated for decades using electrophysiological methods
and many attempts at high-resolution structural profiling of
.alpha.4.beta.2 nAChRs fail due to the exceptionally large (270
amino acids) intracellular domain between transmembrane domains 3
and 4 (hereafter referred to as the M3-M4 loop), which is predicted
from informatics screens to be intrinsically disordered. (FIG. 5).
The inventors used a PDB crystal structure of the Torpedo nicotinic
acetylcholine receptor, used as a proxy for the .alpha.4.beta.2
nicotinic receptor (no structure available) to aid in the
visualization of approximate locations of inter-subunit crosslinks
produced by incubation of live cells with butyldipropriolate and
subsequent receptor purification and analysis with LC-MS/MS. The
inventors used an immobilized mAb299 to immunopurify crosslinked
.alpha.4.beta.2 nAChRs from LTR1-1d cells and interrogate sites of
interaction between subunits in the pentamer using mass
spectrometry. This experiment represents the first successful
application of crosslinking mass spectrometry used to profile sites
of physical interaction between neuronal nAChR subunits. The
inventors identified a number of high-confidence crosslinked
peptides (Table 2a), indicating interactions between various
regions of the .alpha.4 and .beta.2 subunits. Importantly, the
inventors observed two inter-subunit crosslinks occurring between
.alpha.4 and .beta.2 in the large N-terminus and in the entrance to
the "vestibule" of the M3-M4 loop that occupies the region close to
the transmembrane domain (where the peptides exit the lipid
bilayer) (FIG. 8, red circles). This finding represents evidence
that the large intracellular loop regions may closely associate in
proximity to the plasma membrane and play a role in dynamic gating
of the channel upon binding ligand. This example demonstrates the
utility of the crosslinking compounds of this disclosure for the
interrogation and elucidation of specific geometries of interaction
between adjacent intramolecular protein structures.
TABLE-US-00003 TABLE 2a crosslinked peptides of the .alpha.4 and
.beta.2 subunits Linked Residues nAChR Subunit K190-K44
.alpha.4-.alpha.4 K160-K110 .alpha.4-.beta.2 K180-K110
.alpha.4-.beta.2 K361-K342 .alpha.4-.beta.2 K361-K451
.alpha.4-.beta.2
Example 6 Conformational Changes Following Persistent Nicotine
Exposure
[0064] Nicotinic receptors rapidly desensitize following prolonged
exposure to agonist. This functional desensitization is thought to
occur with corresponding changes in the conformation of the
receptor, and .alpha.4.beta.2 nAChRs are particularly prone to
long-lasting periods of functional deactivation following
desensitization by continuous agonist application (Marks et al, J
Neurochem 63(6):2125-35; PMID 7964732; others). The inventors
exposed LTR1-1d cells to nicotine for 24 hours and performed in
vivo crosslinking with butyldipropiolate as described previously to
probe any changes in receptor subunit interactions that may have
occurred as a result of conformational changes associated with
persistent nicotine exposure and desensitization. The inventors
observed differential crosslinking of receptor subunits following
incubation with nicotine compared to those observed under control
conditions (Table 2b). These findings indicate that substantial
shifts in the conformation of .alpha.4.beta.2 nAChRs occur when
ligand is bound that can be observed following crosslinking of
proximal lysine residues. Conformational changes in the receptor
following prolonged agonist occupation when the receptor resides in
the lumen of the endoplasmic reticulum may also facilitate the
binding of interacting proteins that can influence the rate of
receptor assembly and/or transport of nascent receptors
(Srinivassan et al, J Gen Physiol 137(1):59-79; PMID 21187334;
others). This example demonstrates the utility of the crosslinking
compounds of this disclosure for investigating and identifying
dynamic cell signaling events such as agonist-induced
desensitization.
TABLE-US-00004 TABLE 2b crosslinked peptides following incubation
with nicotine Linked Residues nAChR Subunit K44-K95 .beta.2-.beta.2
K99-K103 .alpha.4-.beta.2 K142-K103 .alpha.4-.beta.2 K160-K110
.alpha.4-.beta.2 K180-K110 .alpha.4-.beta.2 K451-K590
.alpha.4-.beta.2
[0065] While the present invention has been illustrated and
described with reference to certain exemplary embodiments, those of
ordinary skill in the art will understand that various
modifications and changes may be made to the described embodiments
without departing from the spirit and scope of the present
invention, as defined in the following claims.
* * * * *