Patent Application
20190358630
The invention relates to thermal shift assays implemented to uncover ligand protein binding interactions in whole cells or cell extracts. In particular embodiments, the invention provides improved methods and devices for performing thermal shift assays for determining both drug targets and drug mechanism of action. For example, the invention performs thermal shift assays in microfluidic devices and determines ligand-protein binding by relative protein abundance measurements.
Conventional thermal shift assays have been used to determine drug interactions using purified samples and cells by detecting thermodynamic stability changes using a variety of methods including western blot and mass spectrometry.
In a typical thermal shift assay, cellular samples are subjected to a thermal shift by using a thermal block originally designed for use in polymerase chain reaction (PCR) amplification. Multiple samples were required at incrementally increasing temperatures in an effort to derive a protein melting curve. The use of thermal blocks is not ideal as they are not designed for thermal shift assays and variations in the heating time and stability can introduce systematic errors. After thermal block heating exposure, the samples next undergo several steps of lysis and centrifugation which involve multiple sample transfers. These steps are time consuming and also introduce a variety of systematic errors which impair subsequent quantitative analysis. In addition, due to the time restrictions current methods are not capable of performing thermal shift assays at short timescales where early drug-protein interactions occur.
For these and other reasons, current methods are limited with respect to accuracy, efficiency, cost-effectiveness, speed and temporal resolution. Accordingly, there is a need in the art for new methods and devices which provide a different measurement strategy for cellular thermal shift assay.
The invention relates to thermal shift assays implemented to uncover ligand protein binding interactions in whole cells or cell extracts. In particular embodiments, the invention provides improved methods and devices for performing thermal shift assays for determining both drug targets and drug mechanism of action. For example, the invention performs thermal shift assays in microfluidic devices and determines ligand-protein binding by relative protein abundance measurements.
In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a microfluidic device comprising at least one microchannel and a plurality of heater elements in contact with said at least one microchannel, and ii) a plurality of cells comprising at least one protein, wherein said at least one protein has a defined melting temperature, iii) a test agent; and b) introducing a first portion of said plurality of cells into a first microchannel; c) introducing second portion of said plurality of cells and said test agent into a second microchannel; c) shifting said temperature in said first and second microchannels to within a defined temperature range with said heating elements; d) measuring a relative protein abundance measurement between said first and second microchannel for said at least one protein; and e) identifying that said protein is bound to said agent by said relative protein abundance measurement. In one embodiment, said relative protein abundance measurement comprises normalized relative protein abundance data. In one embodiment, said shifting of said temperature is performed after a time delay from introducing said second portion of said plurality of cells. In one embodiment, said test agent is a therapeutic agent. In one embodiment, said defined temperature range ranges between 1-3 degrees Celsius of said defined melting temperature of said at least one protein. In one embodiment, said defined temperature range is a defined melting temperature of said at least one protein. In one embodiment, said defined melting temperature ranges between approximately 45-85° C. In one embodiment, said defined melting temperature is 52° C. In one embodiment, said microfluidic device further comprises at least one ultraviolet microchip configured to measure normalize relative protein abundance.
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity but also plural entities and also includes the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
The term “about” or “approximately” as used herein, in the context of any of any assay measurements refers to +/−5% of a given measurement.
As used herein, the term “Tm” is used in reference to a protein “melting temperature.” The melting temperature is the temperature at which a population of isolated proteins denature and/or unfold from a quaternary and/or tertiary conformation into a primary and/or secondary conformation.
The term “channels” or “microchannels” as used herein, refer to pathways (whether straight, curved, single, multiple, in a network, etc.) through a medium (e.g., silicon, glass, polymer, etc.) that allow for movement of liquids and gasses. In some embodiments, described herein, “first channel” and “second channel” are used and these need not have the same shape throughout their length. For example, one can change the channel cross-section, curve or split the channel. Channels can connect or be coupled with other components, i.e., keep components “in communication” and more particularly, “in fluidic communication” and still more particularly, “in liquid communication.” Such components include, but are not limited to, liquid-intake ports and liquid outlet ports. “Microchannels” are channels with dimensions less than 1 millimeter and greater than 1 micron. It is not intended that the present invention be limited to only certain microchannel geometries.
The term “microfluidic” as used herein, relates to components where moving fluid is constrained in or directed through one or more channels wherein one or more dimensions are 1 mm or smaller (microscale). Microfluidic channels may be larger than microscale in one or more directions, though the channel(s) will be on the microscale in at least one direction. In some instances the geometry of a microfluidic channel may be configured to control the fluid flow rate through the channel (e.g. increase channel height to reduce shear). Microfluidic channels can be formed of various geometries to facilitate a wide range of flow rates through the channels.
The term “attached” as used herein, refers to any interaction between a medium (or carrier) and a drug. Attachment may be reversible or irreversible. Such attachment includes, but is not limited to, covalent bonding, ionic bonding, Van der Waals forces or friction, and the like.
The term “drug”, “agent” or “compound” as used herein, refers to any pharmacologically active substance capable of being administered which achieves a desired effect. Drugs or compounds can be synthetic or naturally occurring, non-peptide, proteins or peptides, oligonucleotides or nucleotides, polysaccharides or sugars.
The term “test agent” as used herein, refers to any compound or molecule considered a candidate as a peptide binding ligand.
The term “protein” as used herein, refers to any of numerous naturally occurring extremely complex substances (as an enzyme or antibody) that consist of amino acid residues joined by peptide bonds, contain the elements carbon, hydrogen, nitrogen, oxygen, usually sulfur. In general, a protein comprises amino acids having an order of magnitude within the hundreds.
The term “peptide” as used herein, refers to any of various amides that are derived from two or more amino acids by combination of the amino group of one acid with the carboxyl group of another and are usually obtained by partial hydrolysis of proteins. In general, a peptide comprises amino acids having an order of magnitude with the tens.
The term “polypeptide”, refers to any of various amides that are derived from two or more amino acids by combination of the amino group of one acid with the carboxyl group of another and are usually obtained by partial hydrolysis of proteins. In general, a peptide comprises amino acids having an order of magnitude with the tens or larger.
The term, “purified” or “isolated”, as used herein, may refer to a peptide composition that has been subjected to treatment (i.e., for example, fractionation) to remove various other components, and which composition substantially retains its expressed biological activity.
Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the composition (i.e., for example, weight/weight and/or weight/volume). The term “purified to homogeneity” is used to include compositions that have been purified to “apparent homogeneity” such that there is single protein species (i.e., for example, based upon SDS-PAGE or HPLC analysis). A purified composition is not intended to mean that all trace impurities have been removed.
As used herein, the term “substantially purified” refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and more preferably 90% free from other components with which they are naturally associated. An “isolated polynucleotide” is therefore a substantially purified polynucleotide.
As used herein the term “portion” when in reference to a protein (as in “a portion of a given protein”) refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.
The term “small organic molecule” as used herein, refers to any molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size from approximately 10 Da up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
A “variant” of a protein is defined as an amino acid sequence which differs by one or more amino acids from a polypeptide sequence or any homolog of the polypeptide sequence.
The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have “nonconservative” changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological or immunological activity may be found using computer programs including, but not limited to, DNAStar® software.
In a related aspect, “microfluidic device” is used to describe a substrate comprising at least one channel and may comprise other defined components including surfaces and points of contact between solutions including, but not limited to, reagents, buffers and/or test compounds.
The term “buffer” as used herein, refers to an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small or moderate amount of strong acid or base is added to it and thus it is used to prevent changes in the pH of a solution. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications.
The term “buffer mixture” or “buffer system” as used herein, refers to an aqueous solution consisting of a mixture of at least two weak acids and their conjugate base, or vice versa. The mixture pH changes very little over a wide temperature range (e.g., for example, 1-70° C.) due to buffer-buffer interactions that maintain an overall balanced buffer mixture/system pKa. A buffer mixture/system may also contain other components including, but not limited to, salts, ions, metals and/or supplemental buffer molecules.
The term, “Taylor dispersion” as used herein, refers to an effect in fluid mechanics in which a shear flow can increase the effective diffusivity of a fluid. For example, the shear acts to smear out the concentration distribution within a fluid in the direction of the flow, enhancing the rate at which it spreads in that direction.
The invention relates to thermal shift assays implemented to uncover ligand protein binding interactions in whole cells or cell extracts. In particular embodiments, the invention provides improved methods and devices for performing thermal shift assays for determining both drug targets and drug mechanism of action. For example, the invention performs thermal shift assays in microfluidic devices and determines ligand-protein binding by relative protein abundance measurements.
Comparative melting temperatures (Tm) for evaluating drug binding, e.g. protein kinase drug targets, to protein targets in cancer cell lines using a CETSA method combined with quantitative mass spectrometry have been reported. Cells, or cellular extracts, were treated with one of several drugs including staurosporine. Fold-changes were calculated by using the lowest temperature condition as the reference. Protein fold-changes were normalized. Normalization of data included ‘fraction non-denatured’ (folded/insoluble protein) for identifying a difference in a calculated Tm across the temperature range scan as a change in X (i.e. Delta X). There was no description of normalized protein abundance data determined at or close to a defined protein melting temperature, or use of a microfluidic device. This reference does not describe the use of a microfluidic device to determine relative protein abundance measurement at or within a protein melting temperature to assess ligand protein binding. Savitski, et al. “Tracking cancer drugs in living cells by thermal profiling of the proteome.” Science 346(6205):55 (Summary) and 1255784-1-1255784-10 (Research Article) (2014).
Human non-small cell lung cancer cells have been used in a cellular thermal shift assay (CETSA) combined with analysis by mass-spectrometry of digested proteins' peptides labeled with 10plex Tandem Mass Tags (TMT) for discovering that a drug, i.e. brusatol, regulates Nrf2 activity. More specifically, the melting point of cellular proteins (melting temperature (Tm)) was determined by calculating the temperature from the fitted curve at 50% protein abundance. In fact,
A version of a cellular thermal shift assay using a label-free method for proteome (whole cell) drug target identification combined with in-gel fluorescence difference caused by thermal stability shift (S-FITGE) combined with mass spectrometry has been reported. Western blots were used to confirm identify of the MS identified protein. In contrast with other methods (e.g., quantitative MS-based thermal proteome profiling (TPP) measuring a melting curve shift along the x-axis), this assay revealed a longitudinal Tm comparison based upon deviation along the y-axis between two melting curves. Thermally shifted spots in TS-FITGE 2D electrophoresis of cellular thermal shifted proteins, with and without a drug, were heated and then labeled with Cy2, Cy3, and Cy5 which were considered to be potential target proteins and excised for identification by mass spectrometry. This reference does not describe the use of a microfluidic device to determine relative protein abundance measurement at or within a protein melting temperature to assess ligand protein binding. Park, et al. “Label-free target identification using in-gel fluorescence difference via thermal stability shift” Chem. Sci., 8:1127-1133 (2017).
Cellular thermal shift assay (CETSA) drug target engagement in human cancer cell lines, e.g. K562, mouse cancer cells, mouse livers and mouse kidneys western blot analysis have been described. Multiple aliquots of cell lysate were heated to 9-10 different temperatures with a thermal cycler, between 40° C. and 90° C. After cooling, the samples were centrifuged to separate soluble fractions from precipitated proteins. The presence of the target protein was quantified in the soluble fraction by Western blotting. Relative band intensity (normalized) was graphed against temperature to obtain melting curves, relative to controls, for determining drug target engagement. This reference does not describe the use of a microfluidic device to determine relative protein abundance measurement at or within a protein melting temperature to assess ligand protein binding. Molina, et al., “Monitoring Drug Target Engagement in Cells and Tissues Using the Cellular Thermal Shift Assay” Science 341:84-87 2013.
Cellular thermal shift assays in combination with quantitative mass spectrometry (MS) have been used with denaturing gel electrophoresis and western blot detection using target-specific antibodies, for identifying drug targets the accessible portion of the proteome. Cell lysate, intact cells, biopsies or tissue extracts from a patient, treated or untreated, may provide a sample treated with compounds before heating to at least six temperatures with test compounds added either as a high dose (for melt curve) or, alternatively, as a series of concentrations for a isothermal dose-response fingerprint (ITDRF) CETSA experiment. This reference does not describe the use of a microfluidic device to determine relative protein abundance measurement at or within a protein melting temperature to assess ligand protein binding. Jafari, et al., “The cellular thermal shift assay for evaluating drug target interactions in cells.” Nature Protocols 9(9):2101-2122 (2014).
A cellular thermal shift assay with human cells (K562) heated in a thermocycler and then evaluated by western blotting or mass spectrometry for protein binding to a drug TNP-470 has been described. Differences between the melting curves of corresponding proteins in two patient samples were determined by measuring the differences of soluble protein F for all temperature points. Intensities were normalized and plotted to visualize the changes in melting temperature following ligand treatment. This reference does not describe the use of a microfluidic device to determine relative protein abundance measurement at or within a protein melting temperature to assess ligand protein binding. Norlund et al., “Methods for determining ligand binding to a target protein using a thermal shift assay.” WO 2012/143714.
Thermal shift assay methods have been described for identifying biomarkers (e.g., proteins) indicative of a drug response in a patient by testing treated cells of patients responding to a drug, or before or without a drug treatment, and also compared patients having a reduced response to a drug, analyzed by mass spectrometry. Samples were heated in a PCR thermal block up to a defined temperature for 10 temperatures followed by isobaric labeling for mass spectrometry protein identification. As an example to establish correlations between patient pairs, differences between the melting curves of corresponding proteins in the two patient samples. However, melting curves were graphed comparing relative band intensity (measured from Western blots) to temperature. This reference does not describe the use of a microfluidic device to determine relative protein abundance measurement at or within a protein melting temperature to ligand protein binding. Molina et al., “Method for identifying a biomarker indicative of a reduced drug response using a thermal shift assay” WO 2015/145151.
A microfluidic device with multiple infrared laser heating elements, integrated localized temperature control and integrated optical measurement of fluorescence has been described. The temperature of a single sample can be increased or decreased stepwise using the laser. Fluorescence measurements can be detected for ultraviolet radiation as emitted fluorescence from the samples (especially protein and cells). For example, measurement of the intrinsic fluorescence during thermal excitation enabled the melting temperature (Tm) or mid-point of denaturation (Cm) of the protein to be measured to determine the thermal stability of a protein, across a range of typically 10-50 different temperatures. Thus the effect of additives, bound ligands or drugs, or protein mutations upon the global protein stability is determined. This reference does not describe the use of a microfluidic device to determine relative protein abundance measurement at or within a protein melting temperature to assess ligand protein binding. Aeppli et al., “Microfluidic Device.” WO 2010/149995.
A microfluidic device has been described having one or more resistive heating elements in thermal contact with the at least one portion of the microfluidic channel and a fluorescence detector for providing a thermal melt analysis (Tm) for generating a thermal melting curve using cells. This microfluidic device may be used for receptor-ligand interactions, including those of proteins, for detecting a shift in the thermal denaturation of a protein by a bound molecule. This reference does not describe the use of a microfluidic device to determine relative protein abundance measurement at or within a protein melting temperature to assess ligand protein binding. Sundberg et al., “Method and apparatus for generating thermal melting curves in a microfluidic device.” U.S. Pat. No. 9,376,718 (herein incorporated by reference).
A ligand induced thermal shift assay on a microfluidic device with a heating coil for thermal cycling and a fluorescence detector has been described. The heating coil will typically be disposed adjacent to or in a layer above or below the region of the microchannel wherein heating of the fluid reactants is desired. A shift in the mid-point temperature, the temperature at which the protein is 50% denatured, indicates the protein is bound to a ligand. This reference does not describe the use of a microfluidic device to determine relative protein abundance measurement at or within a protein melting temperature to assess ligand protein binding. Stem S., “Microfluidic devices and methods for performing temperature mediated reactions.” U.S. Pat. No. 6,670,153 (herein incorporated by reference).
Possibilities offered by advances in microfluidic technology for the analysis of therapeutic proteins during manufacturing and formulation have been offered. This reference does not describe the use of a microfluidic device to determine relative protein abundance measurement at or within a protein melting temperature to assess ligand protein binding. Kopp, et al. “Microfluidic approaches for the characterization of therapeutic protein.” J Pharm Sci. Abstract, Jan. 8, 2018.
The Subcellular Pan-Omics for Advanced Rapid Threat Assessment (SPARTA) project has been reported to target the development of a new technological system to rapidly determine how drugs or biological or chemical agents exert their effects on human cells. SPARTA is also developing microfluidic devices to automate sample preparation and control and manipulate individual cell components in order to investigate how protein molecules change in response to a given drug or toxin. As part of the SPARTA project, a microfluidic device was constructed comprising a microchip having dimensions of roughly 3 inches by 1.5 inches. This reference does not describe the use of a microfluidic device to determine relative protein abundance measurement at or within a protein melting temperature to assess ligand protein binding. Anonymous, “National Security: Biomedical innovation in the fast lane.” University of Colorado Boulder Research Report 2014-15.
In one embodiment, the present invention contemplates microfluidic devices including, but not limited to, integrated thermal control electrodes, flow control systems and/or software. In one embodiment, the microfluidic device is configured to perform a thermal shift assay. In some embodiment, the microfluidic devices are capable of sustaining cell growth, determining drug treatment efficacy, performing cell lysis and sample collection. In one embodiment, the microfluidic devices are used for detecting thermodynamic stability changes within cells. In one embodiment, the microfluidic devices are used for validation of drug targets (e.g., a protein drug target). In one embodiment, the microfluidic devices comprise at least one microfluidic channel, wherein the channel contains a plurality of viable cells adhered to the channel surface. In one embodiment, a test agent is introduced into the channel for treatment of the viable cells. In one embodiment, the test agent includes, but is not limited to, a virus, a drug, an antibody and/or a biologically active compound.
The disclosed microfluidic device and methods (microCETSA: MCETSA) are intended as improvements over conventional devices and methods used for thermal shift assays within cells or cell extracts (CETSA). Specifically, as described herein microfluidic devices having multiple heating elements and ultraviolet-based microchips are intended to replace conventional thermal shift assays using PCR thermal blocks coupled with centrifugation. For example, specific disadvantages of conventional thermal shift assays include thermal block introduction of variations in the heating time and stability that result in systematic errors.
The present invention, as contemplated herein solves the many disadvantages of the current state of the art methods using thermal block cycling to determine whether a protein binds a ligand. For example, one popular method constructs a melting temperature profile for all proteins in a cell extract between a vehicle-treated and a drug-treated condition using a thermal block cycling method. Savitski et al., (2014). In particular, it is the shift in the temperature of melting that is of interest (Delta X=Tm(treatment)−Tm(vehicle).
In one embodiment, the present invention contemplates overcoming the above disadvantages by using microfluidic devices. Although it is not necessary to understand the mechanism of an invention, it is believed that one benefit of utilizing a relative protein abundance measurement (e.g., Delta Y), as opposed to Tm shifting (Delta X), is that measurements may be taken at a single defined temperature (e.g., a Tm) or a small temperature range flanking a Tm yields a sufficient statistical difference such that a protein-ligand binding interaction can be identified. In one embodiment, the protein-ligand binding interaction identifies a protein drug target.
Ligand protein binding interactions determined by the presently contemplated relative normalized protein abundance method was compared to the current state of the art method (e.g., Savitski et al. (2014)) in accordance with Example II. Using 52° C. as a single temperature analysis parameter, fifty-seven (57) known drug-protein interactions were identified using the MCETSA method as described herein. Of these fifty-seven (57) targets, Savitski et al. only identified thirty-one (31). Consequently, the Savitski et al. method failed to identify twenty-six (26) of the fifty-seven (57) known drug targets. For example, four (4) of these twenty-six (6) are MAPKAPK2, CSNK2A1, PTMS and RABEP2. See,
A. Microscale Enhanced Cellular Thermal Shift Assay (MCETSA) Methods
The data presented herein show that comparative results between CETSA and MCETSA methods demonstrate greatly improved testing parameters including, but not limited to, decreased ramp time, increased temperature stability, lower drug treatment times; samples required, i.e. fewer samples, and analysis time, i.e. less time, over conventional cellular shift assays.
MCETSA methods can dramatically extend the limitations of current cellular thermal shift assays. Typical parameters of conventional cellular shift assays were compared between CETSA and MCETSA. MCETSA demonstrated dramatic improvements in cell treatment, thermal shift control, automation and decreased sample handling and sample requirements not only improve data quality but decrease costs. See, Table 1.
In one embodiment, the present invention contemplates a method comprising identifying drug binding to a protein. In one embodiment, the protein drug binding is determined by measuring relative protein abundance (Delta Y) at, or close to, a protein's Tm. For example, Delta Y may be determined using a thermal shift at a single temperature or a range of temperatures within 1-3 degrees of each other.
In one embodiment, the present invention contemplates that a MCETSA method that is designed not only to achieve improvements over the art but also have characteristics including, but not limited to:
B. Microscale Enhanced Cellular Thermal Shift Assay (MCETSA) Devices
There are many microscale device configurations that are contemplated by the present invention. Although it is not necessary to understand the mechanism of an invention, it is believed that each of these microscale device configurations can provide the above identified improvements in CETSA. See, Table 1. Some embodiments as contemplated herein comprise characteristics and/or capabilities including but not limited to:
In one embodiment, a microfluidic device further comprises an ultraviolet microchip sensor attached to an outflow channel. In one embodiment, the ultraviolent microchip sensor measures a protein concentration variation across a fluidic interface using an ultraviolet absorption spectra. In one embodiment, the ultraviolet microchip provides, real-time, folded:unfolded protein concentration ratio measurements.
In one embodiment, the present invention contemplates a biologically functional microfluidic device configured to perform a biological protocol. In one embodiment, the biological protocol comprises a microchannel layered with a healthy and viable cell culture. In one embodiment, the cell culture has a cell density of approximately 3.5×105 cells (e.g., for example, spontaneously transformed aneuploid immortal keratinocyte cells; HaCaT cells) within a microchannel. See,
In one embodiment, the present invention contemplates a MCETSA system comprising a microfluidic device (1) attached to an electronics master controller (2), wherein said electronics master controller (2) comprises a fluid switch (3) and a heater electronics board. In one embodiment, the fluid switch (3) is in fluidic communication with an inlet port (4) of the microfluidic device (1). In one embodiment, the fluid switch (3) is in fluidic communication with at least one fluid reservoir (5) containing at least one fluid including, but not limited to, a lysis buffer (e.g., SDS), a serum-free wash buffer (e.g., physiologically buffered saline) and a drug buffer. In one embodiment, the fluid switch (3) is attached to at least one pump (6). In one embodiment, the microfluidic device comprises at least one outlet port (7) for sample collection.
In one embodiment, the present invention contemplates a method of using an MCETSA system comprising: i) seeding cells in a microchannel of a microfluidic device; ii) culturing said cells in said microchannel; iii) attaching said microfluidic device to said master controller; iv) priming tubes and switches in fluidic communication with said microchannel; v) configuring said fluid switch to wash said cell culture approximately 5-10 times; vi) reconfiguring said fluid switch to release a desired cell lysis solution volume; vii) reconfiguring said fluid switch to release a desired drug solution volume; viii) configuring said electronics board to synchronize heater and/or sensor activation with the tail of a drug solution; and ix) collecting a sample from said outlet port.
C. Microscale Enhanced Cellular Thermal Shift Assay (MCETSA) Device Electronics
In some embodiments, the present invention contemplates microscale device configurations comprising electronic control elements. For example, a MCETSA device as contemplated herein may comprise electronic control elements having characteristics and/or capabilities including, but not limited to:
This example demonstrates a current state of the art method to determine ligand protein binding by identifying a protein melting temperature shift. Savitski et al., “Tracking Cancer Drugs in Living Cells by Thermal Profiling of the Proteome” Science 346:6205, 1255784-1-1255784-10 (2014).
Cells were cultured under differential conditions, such as with and without drug treatment. The cells were extracted first, and the extracts were treated with drug (“1”). For each condition, the cell or cell-extract sample was divided into 10 aliquots (“2”). Aliquots were subjected to heating at the indicated temperatures. Samples of intact cells were subsequently subjected to extraction with PBS (“3”). After digestion with trypsin, each sample was labeled with a different TMT10 isotope tag (“4”).
Subsequently, all samples from each condition were mixed (“4”) and analyzed by means of liquid chromatography/mass spectrometry (LC MS/MS (“5”)).
The obtained reporter ion intensities were used to fit a melting curve and calculate the melting temperature Tm of each protein separately for the two conditions (“6”).
This example shows the superior performance of a relative protein abundance method (Delta Y) as compared to a protein melting temperature (Delta X) method to identify protein-ligand interactions.
Briefly, a database of defined protein melting point temperatures was constructed. Savitski et al. (2014). These Tms were determined by: i) using cell extracts (e.g., K562 cell extracts); ii) mixing the cell extracts with either vehicle or staurosporine for ten (10) minutes. Using a standard PCR heating block, the cell extracts were heated for three (3) minutes followed by cooling for three (3) minutes and then subjected to centrifugation. The soluble fraction (e.g., supernatant) was collected and subjected to in-gel digestion. Each individual protein was then bound to a different TMT10 label. The differentially TMT10 labeled proteins were then subjected to RP/RP LC-MS/MS. This protocol identified the Tm for greater than seven thousand proteins (>7,000 proteins).
The Savitski et al. database was used as a source for protein Tm information on which to measure relative normalized abundance data using either a single temperature (e.g., at a specific Tm) or a selected range of temperatures flanking a Tm for a specific protein. The statistical difference between a control measurement and a ligand measurement was calculated using a left-tailed Student's T-test (α=0.01).
The results confirmed that measuring a Delta Y based upon relative normalized protein abundance data successfully identified ligand protein binding interactions when measured by either a single temperature or a small range of selected temperatures flanking a defined temperature (e.g., a protein Tm). Improved identification of all ligand protein interactions within a cell extract (e.g., pepsin digested) was found when three (3) different selected temperatures were used in the analysis. Improved identification of all ligand protein interactions within a cell extract (e.g., pepsin digested) was found at 52° C.
The present application claims the benefit of U.S. Provisional patent Application No. 62/676,013 filed on May 24, 2018, which is incorporated herein by reference.
This invention was made with government support under grant number W911NF-14-2-0019, awarded by the U.S. Army Research Office. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62676013 | May 2018 | US |
