Method, apparatus and computer software for preparing a liquid protein sample for proteome analysis

Abstract

A method for preparing a liquid protein sample for proteomic analysis is provided. The method includes: pumping a liquid protein sample into a heated tube, wherein the liquid protein sample comprises multiple sections; incubating the liquid protein sample in the heated tube, wherein the incubation conditions for a first section of the sample differ from those for a second section of the sample; and pumping the liquid protein sample out of the heated tube.

Description

TECHNICAL FIELDThe present invention relates to the field of proteomics. More particularly, the invention provides an improved method for preparing a liquid protein sample for proteome analysis.PRIOR ARTProteins are linear chains of amino acids and these chains often retain their biological function by folding into compact three-dimensional structures. Since proteins are building blocks of the cell structure and indispensable elements of cell mechanics, most processes taking place in a cell or organism cause changes in three-dimensional structures of the proteins involved in these processes. When cells or isolated proteins interact with other molecules, including other proteins, nucleic acids, metabolites, and drug molecules, the protein structures change. Monitoring these changes is important to understand how cells live, metabolize food, proliferate, communicate with each other, mutate, differentiate and die.In proteomics, it is often desirable to understand how one or more active molecules (e.g., a drug) affect the individual proteins within a proteome.There are a number of prior art methods for analyzing changes in the protein structure of isolated, highly purified proteins, such as thermal shift assay, circular dichroism, hydrogen deuterium mass spectrometry, nuclear magnetic resonance and x-ray crystallography. There are also more recent prior art methods for monitoring structural changes in individual proteins in complex mixtures and at the level of the entire proteome. One such method is Stability of Proteins from Rates of OXidation (SPROX) (West, GM et al. Proc. Natl. Acad. Sci. USA 2010). In this approach, hydrogen peroxide is added to the proteome at varying concentrations to oxidize methionine residues in target proteins. The degree of methionine oxidation depends on the three-dimensional protein structure and is measured in each sample by mass spectrometry. SPROX has been successfully used to identify targets of small molecules such as cyclosporin A. A disadvantage of SPROX is the relatively low frequency of methionine residues in the peptides. A further disadvantage is that hydrogen peroxide causes undesirable side reactions in the protein.Another method for detecting changes in the three-dimensional structure of proteins in non-purified samples is based on limited proteolysis (Piazza, I. et al. Nat. Commun. 2020). 2020). In the method described, the proteome is partially digested by a protease and the digestion rates for various cleavage sites are monitored by proteomics. Changes in the three-dimensional structure of proteins due to different processes result in variations in the cleavage rates of the sites involved in these processes. This method is technically difficult and difficult to automate, as the protein digestion rates depend on a number of experimental parameters which are difficult to control.Thermal Proteome Profiling, TPP (Savitski, MM et al. Science 2014) is a method for examining the protein structure at the proteome level. Figure 1 illustrates a method of thermal proteome profiling (TPP) as described by Savitski et al. The parameter determined in this method is the thermal stability of the protein, which is studied by changing the temperature of the proteome solution from normal (typically 37 degrees Celsius) to increased, for example 67 degrees Celsius or more. At elevated temperatures, many proteins change conformation from natively folded to denatured or fused conformation. Thermal profiling techniques are based on measuring the melting temperature of proteins, which is believed to increase as the protein binds a ligand. The drawback of the thermal profiling methods is that the protein sample must be divided into as many fractions (and reaction vessels) as there are temperature points, typically ten or more points. This approach therefore requires a considerable sample volume for the test procedure and can lead to sample losses (e.g. by manual pipetting). Moreover, since each fraction is incubated at a different temperature, it is a technical challenge to ensure high reproducibility of such analysis.Proteome Integral Solubility Alteration, PISA, (Gaetani, M. et al., J. Proteome Res. 2019, and Sabatier, P., et al., Nat. Commun. 2021, 12, Art No. 6558) provides a method for measuring protein expression and changes in thermal stability in cells. During sample preparation, the cell suspension is distributed over several different PCR tubes and each is heated to a temperature in a specific temperature range. The samples are then combined and protein concentrations measured. Unlike TPP, in PISA, the measured solubility changes are not necessarily interpreted as changes in thermal stability because, as stated in this document, the relationship between solubility and stability is complex.Figure 2 illustrates the PISA method described in this document. In sample preparation, the sample is divided into equal sections which are treated at different temperatures. The samples are then combined into a final mixture for analysis. PISA can therefore be used to reduce the MS analysis time, but still requires several vessels for sample preparation.In a method related to thermal profiling, organic solvents are used instead of temperature for denaturing proteins (Zhang X et al., Anal. Chem. 2020, 92, 1, 1363-1371). Another method uses an increased salt concentration (Beusch C et al., Anal. Chem. 2022, 94, 7066-7074). The advantage of the two processes is that they are isothermal and can be carried out at room temperature. The drawback is that these methods are difficult to automate, while their manual implementation is labor intensive and can lead to variability in the final results. These methods also require a significant amount of sample because the treatment of each fraction is in a different vessel.All existing methods for testing a proteome require numerous manual steps during sample preparation and are therefore error prone and inconsistent. Therefore, improved sample preparation techniques are required for proteome analysis.SUMMARYA method of preparing a liquid protein sample for proteome analysis is provided. The method comprises:pumping a liquid protein sample into a heated tube, the liquid protein sample comprising a plurality of sections;incubating the liquid protein sample in the heated tube, wherein an incubation condition for a first portion of the sample is different from an incubation condition for a second portion of the sample;Pump the liquid protein sample out of the heated hose.Pumping a liquid protein sample into a heated tube may be referred to as "filling" the heated tube. Pumping the liquid protein sample out of the heated tube may be referred to as "eluting" the liquid protein sample.In contrast to the TPP and PISA methods described in the prior art, the invention involves incubating a quantity of sample in a heated tube so that different portions of the sample are exposed to different incubation conditions. As a result, it is no longer necessary to separate the samples for incubation at different temperatures. This maintains the intersample incubation conditions more consistently than manual separation and incubation of samples. The results obtained are therefore more reliable.Moreover, prior art methods requiring manual separation and incubation of samples often require an overall larger amount of sample. Therefore, the method enables more efficient use of the sample.An important difference between the method and the prior art is that the sample sections need not be physically separated into different PCR tubes to meet the required incubation conditions. In contrast to the TTP or PISA methods, one-pot time-induced PISA (OPTIC-PISA) provides different incubation conditions for different sample sections by pumping the sample through a uniformly heated tube at a carefully controlled rate or by subjecting the tube to a temperature gradient. In both cases, the sample prepared by the method is not physically separated and not separately incubated. This is not disclosed in any of the prior art documents, all of which describe that the sample is separated and incubated at different temperatures (and, in the case of PISA, pooled for analysis).The disclosed invention avoids the problems of the prior art methods by performing the incubation in a single vessel, which allows a reduction of the sample volume and a simple automation of the whole incubation process.The assay of the invention may be useful in a number of different fields of biology and medicine. One application is the determination of the protein to which a drug molecule or other ligand binds. Normally, such proteins are referred to as target proteins. In particular in the development of chemical compounds into drugs, it is important to know with which protein the drug interacts. Once the protein target is known, it is important to check whether the compound interacts with the drug target under given conditions and in a given environment. Target identification and monitoring of interactions between target proteins and ligands can therefore be used in initial screening for interacting ligands from large chemical libraries as well as in optimizing an initial ligand for a drug candidate.In other applications in drug development, such as phenotypic screening, in which the desired effect of a drug candidate on live cells or cell lysates is discovered, an important object is to determine which of the thousands of proteins in the cell or lysate interacts with the drug and which protein is thus the target protein for a particular drug.Moreover, it is important to identify the interaction of a drug with other proteins (so-called "off-target interactions") if such interactions can lead to undesirable side effects of treatments. Such side effects may also be desired, in which case the medicament may be used to treat a disease other than that originally intended. Due to the complexity of the processes affecting a drug in a living organism, it is important to verify whether the drug that binds the target protein in a cellular assay also interacts with it in a living organism. It is therefore desirable to be able to detect ligand-protein interactions in complex, non-purified samples, for example body fluids, cell lysates, cells or biopsies of experimental animals or patients. Moreover, it is advantageous if the analytical method is isothermal, can be carried out in a single vessel without sample division and can be automated as far as possible. The present disclosure provides a method of solving this difficulty by performing PISA in a single capillary.In a first embodiment, the tube is heated to a constant temperature. In this embodiment, the method and apparatus are further simplified by performing the incubation at a constant temperature (a single temperature can be provided using a hot plate without complex equipment to establish a temperature gradient). In this embodiment, the different sample sections are incubated at the same temperature but with different incubation times. The pumping rate of the liquid sample is controlled to regulate the incubation time of various portions of the sample.The incubation condition may comprise a period of time during which the respective portion of the sample is incubated in the heated tube.The heated hose can be heated to a predetermined temperature.The entire heated hose can be maintained at a single predetermined temperature.Each of the plurality of sections may be incubated for a different period of time in the heated tube.A fill rate, i.e., a pump rate for pumping the liquid protein sample into the heated tube, may be substantially different from an elution rate, i.e., a pump rate for pumping the liquid protein sample out of the heated tube, such that each of the plurality of sections is incubated for a different period of time in the heated tube.The "velocity" may be expressed as a flow rate in some examples. In other examples, this may be the speed at which the fluid flows through the hose. The flow rate can be calculated by multiplying the velocity by the cross-sectional area of the heated tube.The fill rate and / or elution rate may be varied during filling and / or elution such that each of the plurality of sections is incubated for a different period of time in the heated tube.Incubating the sample in the tube may comprise pumping the sample through the heated tube at a controlled rate such that an incubation time is defined as the time required for each portion of the liquid protein sample to pass from an inlet of the heated tube to an outlet of the heated tube.The controlled speed need not be a constant speed. Since each different portion of the liquid protein sample undergoes a different incubation time, the rate may not be constant. For example, the heated tube may be filled with the sample at a relatively high speed and then the sample may be eluted from the heated tube at a relatively low speed or vice versa.Each of the plurality of sections may be incubated in the heated tube for an average different period of time such that the average incubation time for a first section of the sample is different than the average incubation time for a second section of the sample.In other words, the variation in incubation time for the sample eluting from the tube (sample spatially separated along the length of the tube) may be continuous. Nevertheless, a portion of the sample having a defined volume may be assigned an average incubation time. The average incubation time can be calculated by the integral of the integration time over the volume divided by the volume of the sample.In a second embodiment, a temperature gradient is applied to the tube such that each section is incubated at a different temperature. A second embodiment of the invention involves maintaining a temperature gradient across the heated tube and holding the sample within the tube for a period of time such that the entire sample is incubated for the same period of time but different portions are incubated at different temperatures.The incubation condition may comprise a temperature at which the respective portion of the sample is incubated in the heated tube.The heated tube may be heated to a temperature gradient or a variety of different temperatures.Any different temperature may be applied to a respective portion of the heated tube, wherein each portion of the heated tube may correspond to a portion of the liquid protein sample contained therein.Incubating the sample in the heated tube may comprise keeping the sample stationary in the tube for a predetermined incubation time between the steps of pumping the sample into the tube and pumping the sample out of the tube.The first and second sections may be incubated in the heated tube at different temperatures on average.The sample can be pumped quickly into and out of the heated tube. Nevertheless, the sample may need to pass through each portion of the heated tube to pass from the inlet to the outlet. Therefore, the incubation temperature will not be constant for a particular portion of the sample, since the portion will pass through the temperature gradient / temperatures employed in the heated tube. However, once the heated tube is filled, the sample may be held stationary in the heated tube for a period of time. Therefore, the average incubation temperature for each section approximately corresponds to the temperature applied to that section when the sample is stationary.The incubation conditions may be different for each of the plurality of sections.In other words, not only are the first and second sections subjected to different incubation conditions, but the incubation conditions for each individual section may also be different from the incubation conditions for each other section.The sample may comprise at least three sections, preferably at least five sections, and more preferably about ten sections.A "heated" tube is understood to mean a tube which is actively heated to a temperature above room temperature.The method may further comprise pumping the liquid protein sample into an unheated hose in fluid communication with the heated hose.Pumping the liquid protein sample into the heated hose and / or pumping the liquid protein sample out of the heated hose may include pumping wash solvent into the unheated hose.Because the heated hose and the unheated hose are in fluid communication, pumping wash solvent into the unheated hose may cause the liquid protein sample in the unheated hose to be forced out of the unheated hose and into the heated hose. Pumping further wash solvent into the unheated hose may cause the wash solvent in the unheated hose to be forced out of the unheated hose into the heated hose, thereby forcing the liquid protein solution out of the heated hose.The method may further comprise creating an air bubble in the unheated tubing between the liquid protein sample and the wash solvent.The air bubble may keep the wash solvent separate from the liquid protein sample to isolate the liquid protein sample and prevent contamination.The method may further comprise cooling the liquid protein sample after it is pumped from the heated tube. By cooling the liquid protein sample, it can be stabilized, so that further heat-induced changes of the proteins in the sample are limited.The method may further comprise analyzing the liquid protein sample to determine a relative frequency of one or more proteins in the sample.The liquid protein sample can be analyzed using an analyzer, such as a mass spectrometer.Preferably, the portions of the treated sample are analyzed together (in a PISA procedure). Alternatively, the different portions of the sample could be separated into different fractions and analyzed separately (in a TPP method).Pumping the liquid protein sample from the heated tube may include pumping the liquid protein sample from the heated tube in a plurality of fractions.Each of the plurality of fractions may correspond to a portion of the liquid protein sample such that an incubation condition for a first fraction of the sample is different from an incubation condition for a second fraction of the sample.The method may further comprise separately analyzing each fraction of the liquid protein sample to determine the relative frequency of one or more proteins in each fraction.The liquid protein sample may comprise a protein mixture and / or live cells.The method may further comprise adding one or more test compounds to the liquid protein sample prior to incubating the liquid protein sample in the heated tube.An apparatus configured to perform any of the methods described above is provided. The apparatus comprises:a heated hose,a pump configured to pump a liquid protein sample into a heated tube and pump the liquid protein sample out of the heated tube.The apparatus may further comprise one or more of the following elements:a fraction collector configured to collect the liquid protein sample pumped from the heated tube; andan analyzer configured to analyze the liquid protein sample pumped from the heated tube.Computer software is also provided. The computer software includes instructions that, when executed on a processor, cause the processor to perform a method as described above.Further examples useful for understanding the invention are set forth in the following numbered paragraphs:Paragraph 1. A method for detecting target proteins for a compound or compounds added to an unpurified complex protein mixture, comprising: (a) preparing a liquid sample consisting of a protein mixture or living cells with or without test compound(s) added; (b) filling a tube, which is entirely or partially maintained at a fixed temperature, with the sample; (c) eluting the sample from the tube after a period of from zero to several minutes.Paragraph 2 The method of Paragraph 1, wherein the elution rate of the sample is substantially different from the filling rate such that different volumes of the sample are exposed to that temperature for different time intervals.Paragraph 3 The method of Paragraph 1, wherein the filling rate or the elution rate or both rates are varied during filling or elution so that different volumes of sample are exposed to that temperature for different time intervals.Paragraph 4 Apparatus using the method of Paragraph 1 and comprising at least the following modules: an autosampler, a pump, a tube maintained at constant temperature, and a fraction collector, wherein the autosampler contains protein samples and the pump allows the tubes to be filled with protein samples and eluted from the tubes into the fraction collector.Paragraph 5 The apparatus of Paragraph 4 wherein all said modules are programmable in the software or user interface and all operating steps are programmed prior to performing samples in the software or user interface.In addition, an instrument for automatically carrying out the methods of this invention will be described.BRIEF DESCRIPTION OF THE DRAWINGSFigure 1 illustrates a method for thermal proteome profiling (TPP). Figure 2 illustrates a method for altering the integral solubility of proteomes (PISA). FIG. 3A illustrates an example device and the flow of a liquid protein sample through the example device. FIG. 3B illustrates a plot of the flow rate of a liquid protein sample over time for the device illustrated in FIG. 3A. FIG. 4 illustrates a plot of the incubation time for a portion of the sample versus the position of the sample in the tubing for the device of FIG. 3A and the flow rate of FIG. 3B. FIG. 5A illustrates another example device. FIG. 5B illustrates a plot of the flow rate of a liquid protein sample over time for the device illustrated in FIG. 5A. FIG. 6 illustrates results of a specific example method.DETAILED DESCRIPTIONOne method of understanding the effects of one or more active molecules (e.g., a drug) on the individual proteins within a proteome is thermal proteome profiling (TPP), which relies on changes in the thermal stability of proteins to determine how the molecule(s) affect the various proteins within a proteome. As described in Savitski et al., this includes:dividing a sample into many fractions,heating each fraction to a different temperature (in some examples, 10 different fractions are used for each of 10 different temperatures), andDetermine a "melting curve" for each protein in the sample.A melting curve is a plot of protein frequency versus temperature (see Figure 1).Typically, the melting curves of "target" proteins that bind to the molecule(s) are shifted towards higher temperatures than those of proteins that do not bind to the molecule(s). This can be done, for example, because the thermal stability of the protein increases when it is bound to the molecule(s).A related method is the Proteome Integral Solubility Alteration (PISA) assay, examples of which are described in Gaetani et al. and Sabataier et al., which is an alternative to TPP, where the samples can be analyzed together. This may possibly improve the throughput of the proteome profiling process.Some example methods allow for the analysis of changes in the three-dimensional structure of proteins by providing a differential solubility assay in which two or more proteome states are compared for the differences in the solubility of individual proteins in these states. Since most proteins are present in the cell in both soluble and insoluble states (Vecchi et al., PNAS 2020, 117, 1015-1020), almost all known proteomic protocols involve the step of separating the soluble and insoluble portions of the proteome. Most approaches to proteomics are based on measuring the frequencies of soluble proteins as a measure of the protein concentration in the sample. The frequency of soluble proteins can be used as a measure of protein solubility in a particular proteome state.To ensure that not all molecules of a particular protein are fully soluble in each proteome state, and thus to increase the dynamic range of solubility measurements, a portion of the sample is incubated at a temperature other than ambient temperature. The solubility of many proteins decreases at higher temperatures, but some proteins lose their solubility at a temperature near freezing. Moreover, related proteins may have different solubility characteristics when derived from different organisms. Therefore, the optimum incubation temperature will depend on the specifics of the proteins of interest.The invention describes a method and a device for the commercial automation of the TPP or PISA method. The original TPP and PISA methods involve dispensing a sample into many (e.g., about 10) fractions and incubating each fraction at a different temperature. This is a relatively complex, time-consuming manual task and requires a relatively large amount of sample (e.g., ~10x ~10μL).In the TPP and PISA methods of the prior art, incubation is at different temperatures but over the same period of time. In contrast, the invention obtains the same (or similar) information using only one sample set ("one-pot" method) in a manner that is substantially less complex and suitable for automation.In some examples, the method is not based on multiple different temperatures, but on different incubation times. The incubation time is different for different portions of the liquid protein sample. In such examples, the incubation temperature may be a fixed temperature.To achieve this, the volume in which the incubation takes place is spatially extended, such as in a tube or capillary, and different portions of the sample are exposed to a fixed temperature for different periods of time.FIG. 3A illustrates an example apparatus and flow of a liquid protein sample through the heated tube to implement some example methods. FIG. 3B illustrates a plot of the flow rate of a liquid protein sample over time for some example methods. Figure 4 illustrates a plot of the incubation time for a portion of the sample versus the position of the sample in the tubing.Another example device is illustrated in FIG. 5A. FIG. 5B illustrates a plot of the flow rate of a liquid protein sample over time for other example methods.In a first example, a sample is loaded into a long capillary tube that is heated to a fixed temperature, e.g., by being attached to a hot plate ("hot coil"). The rates at which the sample is loaded into and removed (eluted) from the tube may be controlled separately by one or more pumps. The sample is loaded into the heated tube for a relatively short period of time (e.g., ~10 sec). The sample is then eluted from the heated tube over a relatively long period of time (e.g., over a period of ~10 minutes). The portions of the sample eluted relatively early in the experiment from the heated tube will have been incubated for a short period of time, while the portions of the sample eluted relatively late in the experiment from the heated tube will have been incubated for a long period of time. By analyzing the various portions of the sample that have undergone different incubation times, "melting curves" for each protein in the sample can then be obtained.The method has been shown to be automatable and to work well with small sample amounts (e.g., ~0.9 μg).In a second example, a long capillary tube could be attached to a hot plate with two (or more) heating elements maintained at different temperatures so that a temperature gradient is maintained across the plate / capillary. All sample could be retained in the capillary for the same period of time. Due to the temperature gradient, different portions of the sample within the capillary would be exposed to different temperatures.Although the frequency of each protein is preferably measured by mass spectrometry, other techniques could be used, in principle or instead, such as western blotting, mass photometry, etc.A known problem with this method is cross-contamination of samples (since the sample adheres to the walls of the tube). This can be addressed by washing and / or passivating the tube prior to each measurement.Instead of a single heating plate, two heating plates could also be used, between which the tube is held, for example. Alternatively, a heating wire could be wound around the tube.In a specific example, an apparatus comprises: A) an autosampler having input wells with liquid samples of protein mixtures or living cells and a needle from which defined volumes of this liquid sample can be taken. The autosampler may have a cooling system that keeps the sample stable by cooling in the rest of the device prior to processing. B) A pump capable of transferring said volumes from the needle into a tube maintained at a constant temperature, for example, by being attached to a hot plate made of a material having a high thermal capacity, such as metal or ceramic. It is advantageous to produce the tube also from materials with a high heat capacity, such as quartz glass. C) AN electronic temperature control system capable of maintaining a fixed temperature of the plate that is elevated compared to the ambient temperature. D) A fraction collector capable of receiving liquid sample volumes eluted through the tubing and collecting them into dispensing wells. E) A software program for operating the needle, pump, control system and fraction collector.A specific example method includes the following steps: a) Preparation step: Preparation of liquid samples containing cell lysates or intact cells with or without the molecule of interest, e.g., a drug, and placing the prepared sample in a particular input well of the autosampler, b) Treatment step: using the pump, transferring the liquid sample from a particular input well into a tube, and then eluting the liquid sample from the tube into the fraction collector such that different sample volumes are exposed to the temperature maintained in the tube for different periods of time. This can be achieved either by a different constant fill rate into the tube compared to the constant elution rate from the tube or by using a different fill rate into the tube or by using a different elution rate from the tube or by a combination thereof. c) Collection step: With the fraction collector, the eluted sample is placed in an output well corresponding to the input well from which the respective sample originates. d) Lysis step (for samples only, The present invention provides a method for purifying proteins containing intact cells by using quantitative proteomics. g) Data processing step: comparing relative protein frequencies and determining proteins whose frequency and thus their solubility has changed in the presence of a drug.Some example methods can be used in the analysis of single proteins and protein mixtures, including proteins in unfractionated cell lysates and intact cells. Other example methods may be used in determining changes in protein solubility due to thermal unfolding. One application is the determination of proteins in an unpurified complex sample that interact with a test compound of interest added to the mixture.The result of the differential solubility assay is, in the simplest case, the difference or ratio of the frequencies of these two states for each protein. Such measurements performed in a series of replicates provide the statistical significance of the difference or ratio via a suitable statistical test, such as the Student's t-test.FIG. 6 illustrates the results of a specific example. Cell lysate was treated with methotrexate (MTX) and with a vehicle (DMSO) for control. 5 μl samples in 5 replicates were placed in an autosampler. Using an automatic syringe, 3 μl samples were sequentially loaded into an OPTI-PISA capillary at high speed (<1 min). After a short time, the samples were slowly eluted from the OPTI-PISA capillary into a fraction collector (≥10 min). The collected samples were digested, TMT labeled, and analyzed by LC-MS / MS. The MTX target DHFR was identified as outliers in a vulcanic diagram with excellent p-value (<10 -8). Only 900 ng of lysate was used per sample. The results indicate a better p-value than in a corresponding manual PISA experiment. The improvement may be due, at least in part, to the automation of sample incubation.The described OPTI-PISA methods therefore provide a number of advantages. Some methods allow for the complete automation of heat treatment for PISA assays on lysates. In some embodiments, sample consumption is low over prior art methods and is below 1 μg per sample in some examples. Some example methods therefore offer a high reproducibility of the results on account of reliable automation. The results could therefore provide a better (lower) p-value than manual PISA trials with the same samples. In some example methods, the treatment time per sample may be about 10-15 minutes. Therefore, the described methods provide a high throughput compared to prior art methods. The methods described may also be compatible with automated proteome analysis equipment and software.In describing the present invention, it should be understood that a word used in the singular also includes its plural counterpart and a word used in the plural also includes its singular counterpart unless otherwise implicitly or explicitly understood or indicated. Furthermore, it should be understood that for any given component or embodiment described herein, in general, any of the possible candidates or alternatives listed for that component may be used alone or in combination with one another, unless otherwise implicitly or explicitly understood or indicated. Moreover, it should be appreciated that the figures shown herein are not necessarily to scale, and some of the elements may be drawn merely to explain the invention. Also, reference numerals may be repeated among the various figures to represent corresponding or analogous elements. Moreover, it should be understood that each list of such candidates or alternatives is intended for purposes of illustration only and is not intended to be limiting unless otherwise implied or explicitly understood or indicated.Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. In conflict, the present specification, including definitions, applies. It should be understood that prior to the quantitative terms mentioned in the present specification, there is an implicit "about" such that minor and insubstantial deviations are within the scope of the present teachings. In this application, the use of the singular includes the plural, unless expressly stated otherwise. The use of "comprise," "comprises," "comprising," "include," "includes," "containing," "include," "including," "including," and "includes" is not intended to be limiting. As used herein, "a" or "an" may also refer to "at least one" or "one or more.". Further, the use of "or" is inclusive such that the term "A or B" is true when "A" is true, "B" is true, or both "A" and "B" are true.References included in the specificationThis list of documents cited by the applicant has been produced in an automated manner and is only included for the better information of the reader. The list is not part of the German patent application or utility model application. The DPMA does not take any adhesion for any faults or omissions.Cited Non-Patent LiteratureWest, GM et al. Proc. Natl. Acad. Sci. USA 2010

[0004] Piazza, I. et al. Nat. Commun. 2020

[0005] Thermal Proteome Profiling, TPP (Savitski, MM et al. Science 2014

[0006] Gaetani, M. et al., J. Proteome Res. 2019

[0007] Sabatier, P, et al. Nat. Commun. 2021, 12

[0007] Zhang X et al., Anal. Chem. 2020, 92, 1, 1363-1371

[0009] Beusch C et al., Anal. Chem. 2022, 94, 7066-7074

[0009] Vecchi et al., PNAS 2020, 117, 1015-1020

[0066]

Claims

A method of preparing a liquid protein sample for proteome analysis, the method comprising: pumping a liquid protein sample into a heated tube, wherein the liquid protein sample comprises a plurality of sections; incubating the liquid protein sample in the heated tube, wherein an incubation condition for a first section of the sample is different from an incubation condition for a second section of the sample; pumping the liquid protein sample out of the heated tube.The method of claim 1, wherein the incubation condition comprises a period of time during which the respective portion of the sample is incubated in the heated tube.The method of claim 1 or 2, wherein the heated hose is heated to a predetermined temperature.The method of any preceding claim, wherein each of the plurality of sections is incubated for a different period of time in the heated tube.The method of any preceding claim, wherein a fill rate, which is a pumping rate for pumping the liquid protein sample into the heated tube, is substantially different than an elution rate, which is a pumping rate for pumping the liquid protein sample out of the heated tube.The method of claim 5, wherein the filling rate and / or elution rate are varied during filling and / or elution such that each of the plurality of sections is incubated for a different period of time in the heated tube.The method of any preceding claim, wherein incubating the sample in the tubing comprises pumping the sample through the heated tubing at a controlled rate such that an incubation time is defined as the time required for the respective portion of the liquid protein sample to pass from an inlet of the heated tubing to an outlet of the heated tubing.The method of any preceding claim, wherein each of the plurality of sections is incubated in the heated tube for an average different period of time such that the average incubation time for a first section of the sample is different from the average incubation time for a second section of the sample.The method of claim 1, wherein the incubation condition comprises a temperature at which the respective portion of the sample is incubated in the heated tube.The method of claim 9, wherein the heated hose is heated to a temperature gradient or a plurality of different temperatures.The method of claim 9 or claim 10, wherein incubating the sample in the heated tube comprises maintaining the sample stationary in the tube for a predetermined incubation period between the steps of pumping the sample into the tube and pumping the sample out of the tube.The method of any one of claims 9 to 11, wherein the first and second portions are incubated in the heated tube at different temperatures on average.The method of any preceding claim, wherein the incubation condition is different for each of the plurality of sections.The method of any preceding claim, further comprising pumping the liquid protein sample into an unheated hose in fluid communication with the heated hose.The method of claim 14, wherein pumping the liquid protein sample into the heated hose and / or pumping the liquid protein sample out of the heated hose comprises pumping a washing solvent into the unheated hose.The method of claim 15, further comprising creating an air bubble in the unheated tubing between the liquid protein sample and the wash solvent.The method of any preceding claim, further comprising cooling the liquid protein sample after it has been pumped from the heated tube.The method of any preceding claim, further comprising analyzing the liquid protein sample to determine a relative frequency of one or more proteins in the sample.The method of any one of claims 1 to 17, wherein pumping the liquid protein sample from the heated tube comprises pumping the liquid protein sample from the heated tube in a plurality of fractions.The method of claim 19, further comprising separately analyzing each fraction of the liquid protein sample to determine the relative frequency of one or more proteins in each fraction.The method of any preceding claim, wherein the liquid protein sample comprises a protein mixture and / or live cells.The method of any preceding claim, further comprising adding one or more test compounds to the liquid protein sample prior to incubating the liquid protein sample in the heated tube.An apparatus configured to perform the method of any one of claims 1 to 22, the apparatus comprising: a heated hose; a pump configured to pump a liquid protein sample into a heated hose and pump the liquid protein sample out of the heated hose.The apparatus of claim 23, further comprising one or more of: a fraction collector configured to collect the liquid protein sample pumped from the heated tube; and an analyzer configured to analyze the liquid protein sample pumped from the heated tube.Computer software comprising instructions which, when executed on a processor, cause the processor to perform a method according to any one of claims 1 to 22.

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