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

Abstract

A method of preparing a liquid protein sample for proteomic analysis provides a method of preparing a liquid protein sample for proteomic analysis. The method comprises: pumping a liquid protein sample into a heating tube, wherein the liquid protein sample comprises a plurality of portions; incubating the liquid protein sample in the heating tube, wherein incubation conditions of a first portion of the sample are different from incubation conditions of a second portion of the sample; and pumping the liquid protein sample out of the heating tube.

Description

Technical Field

[0001] The present invention relates to the field of proteomics. In particular, the present invention provides an improved method for preparing liquid protein samples for proteomic analysis. Background Art

[0002] Proteins are linear chains of amino acids, and these chains usually obtain their biological functions when they are folded into compact three-dimensional structures. Since proteins are building blocks of cell structure and indispensable elements of cell mechanics, most processes occurring in cells or organisms induce changes in the three-dimensional structures of proteins involved in these processes. When cells or isolated proteins interact with other molecules (including other proteins, nucleic acids, metabolites, and drug molecules), protein structure changes. Monitoring these changes is important for understanding how cells survive, metabolize food, proliferate, communicate with each other, mutate, differentiate, and die.

[0003] In proteomics, one often wishes to understand how one or more active molecules (eg, drugs) affect each of the different proteins within the proteome.

[0004] For isolated, highly purified proteins, there are many prior art methods for analyzing protein structural changes, such as thermal shift determination, circular dichroism, hydrogen-deuterium mass spectrometry, nuclear magnetic resonance and X-ray crystallography. There are also some recent prior art methods to monitor structural changes of individual proteins in complex mixtures and structural changes at the level of the entire proteome. One such method is protein stability from oxidation rate (SPROX) (West, GM et al. Proc. Natl. Acad. Sci. USA 2010). In this method, hydrogen peroxide is added to the proteome at different concentrations to oxidize methionine residues in the target protein. The methionine oxidation level depends on the three-dimensional protein structure and is measured by mass spectrometry in each sample. SPROX has been successfully used to identify targets of small molecules, such as cyclosporin A. One disadvantage of SPROX is that the frequency of methionine residues in peptides is relatively low. Another disadvantage is that hydrogen peroxide induces undesirable side reactions in proteins.

[0005] Another method for detecting changes in protein three-dimensional structure in unpurified samples is based on limited proteolysis (Piazza, I. et al. Nat. Commun. 2020). In the described method, the proteome is partially digested by proteases, and the digestion rates of different cleavage sites are monitored by proteomics. Changes in the three-dimensional structure of proteins caused by different processes induce changes in the cleavage rates of sites involved in these processes. This method is technically difficult and difficult to automate because the protein digestion rate depends on many experimental parameters that are difficult to control.

[0006] Thermal Proteome Profiling (TPP) (Savitski, MM et al. Science 2014) is a method for probing protein structure at the proteome level. Figure 1 Thermoproteome analysis (TPP) methods are shown, as described by Savitski et al. The parameter evaluated in this method is protein thermal stability, which is detected by changing the temperature of the proteome solution from a normal temperature (usually 37 degrees Celsius) to an elevated temperature (such as 67 degrees Celsius or higher). At elevated temperatures, many proteins change their conformation from native folding to a denatured or molten conformation. Thermal analysis methods are based on measuring protein melting temperatures, and it is speculated that the melting temperature increases when the protein binds to a ligand. The disadvantage of thermal analysis methods is that they need to divide the protein sample into as many fractions (and reaction vessels) as temperature points, typically ten or more points. Therefore, this method requires a large amount of samples for testing and may cause sample loss (e.g., by manual pipetting). In addition, since each fraction is incubated at different temperatures, it is technically challenging to ensure the high reproducibility of this analysis.

[0007] Proteome Integral Solubility Alteration (PISA) (Gaetani, M. et al. J. Proteome Res. 2019 and Sabatier, P, et al. Nat. Commun. 2021, 12, art.nr. 6558) provides a method for measuring changes in protein expression and thermal stability in cells. During sample preparation, the cell suspension is distributed into multiple different PCR tubes, and each PCR tube is heated to a temperature within a given temperature range. The samples are then merged together and the protein concentration is measured. Unlike TPP, in PISA, the measured solubility changes are not necessarily interpreted as thermal stability changes, because as described in this document, the relationship between solubility and stability is complex.

[0008] Figure 2 The PISA method described in this document is shown. During sample preparation, the sample is divided into equal parts for processing at different temperatures. The samples are subsequently pooled in the final mixture for analysis. Therefore, PISA can be used to reduce MS analysis time, but still requires multiple containers for sample processing.

[0009] A method involving thermal analysis uses an organic solvent instead of temperature to denature proteins (Zhang X et al., Anal. Chem. 2020, 92, 1, 1363-1371). Another method uses increased salt concentration (Beusch C et al., Anal. Chem. 2022, 94, 7066-7074). The advantages of these two methods are that they are isothermal and can be performed at room temperature. The disadvantage is that these methods are difficult to automate, and their manual implementation is labor-intensive and may lead to variability in the final result. These methods also require a large amount of samples because the processing of each fraction is carried out in different containers.

[0010] Existing methods for probing the proteome all require extensive manual steps during sample preparation and are therefore prone to errors and inconsistencies. Therefore, there is a need for an improved method for preparing samples for proteome analysis. Summary of the invention

[0011] A method for preparing a liquid protein sample for proteome analysis is provided. The method comprises:

[0012] A liquid protein sample is pumped into a heated tube, wherein the liquid protein sample comprises a plurality of

[0013] part;

[0014] incubating the liquid protein sample in the heated tube, wherein the incubation conditions for a first portion of the sample are different than the incubation conditions for a second portion of the sample;

[0015] The liquid protein sample is pumped out of the heated tube.

[0016] Pumping the liquid protein sample into the 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.

[0017] Compared to the TPP and PISA methods described in the prior art, the present invention incubates an amount of sample in a heated tube so that different parts of the sample are subjected to different incubation conditions. This eliminates the need to separate samples for incubation at different temperatures. Therefore, compared to manual separation and incubation of samples, incubation conditions are applied more consistently between samples. Therefore, the results obtained are more reliable.

[0018] Furthermore, prior art methods that require manual separation and incubation of samples generally require larger amounts of sample overall. Therefore, this method enables more efficient use of the sample.

[0019] An important difference between the method and the prior art is that, in order to achieve required incubation conditions, the parts of the sample do not need to be physically separated into different PCR tubes. Different from TTP or PISA methods, one pot time induction (OPTI)-PISA provides different incubation conditions for different parts of the sample by pumping the sample through a uniform heating tube or by subjecting the tube to a temperature gradient at a carefully controlled speed. In either case, the sample prepared by the method is not physically separated and is not incubated separately. This is not disclosed in any prior art document, and the prior art document describes that the sample is separated and incubated at different temperatures (and, in the case of PISA, is brought together for analysis).

[0020] The disclosed invention avoids the problems of prior art methods by performing the incubation in a single vessel, thereby allowing for sample volume reduction and easy automation of the entire incubation process.

[0021] The assay of the present invention can be used in several different biological and medical fields. One application is to determine the protein to which a drug molecule or another ligand binds. Typically, such proteins are referred to as target proteins. In particular, in the process of developing chemical compounds into drugs, it is important to know which protein the drug interacts with. When the protein target is known, it is important to verify that the compound interacts with the drug target under given conditions and in a given environment. Therefore, target identification and monitoring of target protein-ligand interactions can be used for initial screening of interacting ligands from large chemical libraries, and for optimizing initial ligands into candidate drugs.

[0022] In other drug development applications, such as phenotypic screening, where the desired effect of a drug candidate is discovered on living cells or cell lysates, an important task is to determine which of the thousands of proteins in the cells or lysates interact with the drug and therefore determine which protein is the target protein for a given drug.

[0023] In addition, it is important to identify interactions of drugs with other proteins (so-called "off-target interactions"), where such interactions may lead to undesirable therapeutic side effects. Such side effects may also be desirable, in which case the drug can be repurposed to treat a different condition than originally planned.

[0024] Due to the complexity of the process that affects medicine in living organisms, it is important to verify that the medicine in conjunction with target protein in cell assay also interacts with the target protein in living organisms. Therefore, it is desirable to detect the ligand-protein interaction in complicated unpurified samples, such as body fluid, cell lysate, cell or biopsy from experimental animals or patients. In addition, it is advantageous to make analytical method isothermal, carry out in single container without sample separation and automation as far as possible. The invention provides the method for solving this difficulty by carrying out PISA in single capillary.

[0025] In a first embodiment, the tube is heated to a constant temperature. In this embodiment, the method and apparatus are further simplified by incubating at a constant temperature (a heating plate can be used to provide a single temperature without the need for complex equipment to establish a temperature gradient). In this embodiment, different parts of the sample are incubated at the same temperature, but with different incubation times. The pumping speed of the liquid sample is controlled to adjust the incubation time of different parts of the sample.

[0026] The incubation conditions may include a period of time for incubating the corresponding portion of the sample in the heated tube.

[0027] The heating tube may be heated to a predetermined temperature.

[0028] The entire heating tube can be maintained at a single predetermined temperature.

[0029] Each of the multiple portions may be incubated in the heated tube for a different period of time.

[0030] The fill rate, which is the pumping rate used to pump the liquid protein sample into the heated tube, can be significantly different from the elution rate, which is the pumping rate used to pump the liquid protein sample out of the heated tube, so that each of the multiple parts is incubated in the heated tube for a different period of time.

[0031] In some examples, "speed" can be expressed as flow rate. In other examples, this can be the speed at which the fluid travels along the tube. The flow rate can be calculated as the speed multiplied by the cross-sectional area of ​​the heating tube.

[0032] The filling speed and / or elution speed can be changed during filling and / or elution so that each of the multiple parts is incubated in the heated tube for a different period of time.

[0033] Incubating the sample in the tube may include pumping the sample through the heated tube at a controlled rate such that an incubation period is defined as the time it takes for a corresponding portion of the liquid protein sample to travel from an inlet of the heated tube to an outlet of the heated tube.

[0034] The controlled speed need not be a constant speed. In fact, in order to allow each different portion of the liquid protein sample to undergo a different incubation period, the speed may not be constant. For example, the heated tube may be filled with 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.

[0035] Each of the plurality of portions may be incubated in the heated tube for a different average time period such that an average incubation time for a first portion of the sample is different than an average incubation time for a second portion of the sample.

[0036] In other words, the variation of the incubation time of samples eluted from the tube (samples are spatially separated along the length of the tube) can be continuous. However, a portion of the sample having a defined volume can have an associated average incubation time. The average incubation time can be calculated by integrating the integral time over the volume and dividing by the volume of the sample.

[0037] In a second embodiment, a temperature gradient is applied to the tube so that each portion is incubated at a different temperature.A second embodiment of the invention involves maintaining a temperature gradient at the heated tube and holding the sample within the tube for a period of time so that all samples are incubated for the same amount of time, but different portions are incubated at different temperatures.

[0038] The incubation conditions may include the temperature at which the corresponding portion of the sample is incubated in the heated tube.

[0039] The heating tube can be heated to a temperature gradient or to multiple different temperatures.

[0040] Each different temperature may be applied to a corresponding portion of the heated tube, and each portion of the heated tube may correspond to a portion of the liquid protein sample contained therein.

[0041] Incubating the sample in the heated tube may include holding 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.

[0042] The first part and the second part can be incubated in a heating tube at average different temperatures.

[0043] The sample can be pumped in and out of the heating tube quickly. However, the sample may need to travel through each section of the heating tube to reach the outlet from the inlet. Therefore, as a given section of the sample passes through the temperature gradient / different temperatures applied by the heating tube, the incubation temperature of that section will not be constant. However, once the heating tube has been filled, the sample can remain stationary in the heating tube for a period of time. Therefore, when the sample is stationary, the average incubation temperature of each section will be approximately equal to the temperature applied to that section.

[0044] The incubation conditions may be different for each of the multiple portions.

[0045] In other words, in addition to the first portion and the second portion being subjected to different incubation conditions, the incubation conditions of each portion may be different from the incubation conditions of each other portion.

[0046] The sample may comprise at least three portions, preferably at least five portions, more preferably about ten portions.

[0047] A "heated" tube may be construed as a tube that is actively heated above room temperature.

[0048] The method may further include pumping the liquid protein sample into an unheated tube in fluid communication with the heated tube.

[0049] Pumping the liquid protein sample into the heated tube and / or pumping the liquid protein sample out of the heated tube may include pumping a wash solvent into an unheated tube.

[0050] Since the heated and unheated tubes are in fluid communication, pumping wash solvent into the unheated tube may cause the liquid protein sample in the unheated tube to be pushed out of the unheated tube and into the heated tube. Pumping additional wash solvent into the unheated tube may cause the wash solvent in the unheated tube to be pushed out of the unheated tube and into the heated tube, thereby pushing the liquid protein solution out of the heated tube.

[0051] The method may further include generating bubbles in the unheated tube between the liquid protein sample and the wash solvent.

[0052] The air bubbles can keep the washing solvent separated from the liquid protein sample to separate the liquid protein sample and prevent contamination.

[0053] The method may further comprise cooling the liquid protein sample after it has been pumped out of the heated tube.Cooling the liquid protein sample may help to stabilize it so that further heat-induced changes to the proteins in the sample are limited.

[0054] The method may also include analyzing the liquid protein sample to determine the relative abundance of one or more proteins in the sample.

[0055] Liquid protein samples can be analyzed using analytical instruments such as a mass spectrometer.

[0056] Preferably, the parts of the processed sample are analyzed together (in the PISA method). Alternatively, different parts of the sample can be separated into different fractions and analyzed separately (in the TPP method).

[0057] Pumping the liquid protein sample out of the heated tube may include pumping the liquid protein sample out of the heated tube in a plurality of fractions.

[0058] Each fraction of the plurality of fractions may correspond to a portion of the liquid protein sample, such that incubation conditions of a first fraction of the sample are different than incubation conditions of a second fraction of the sample.

[0059] The method may also include analyzing each fraction of the liquid protein sample individually to determine the relative abundance of one or more proteins in each fraction.

[0060] The liquid protein sample may contain a protein mixture and / or living cells.

[0061] The method may further include adding one or more test compounds to the liquid protein sample prior to incubating the liquid protein sample in the heated tube.

[0062] A device configured to perform the above method is also provided. The device comprises:

[0063] Heating tube,

[0064] A pump is configured to pump the liquid protein sample into the heated tube and to pump the liquid protein sample out of the heated tube.

[0065] The device may also include one or more of the following:

[0066] a fraction collector configured to collect the liquid protein sample pumped out of the heated tube, and

[0067] An analytical instrument is configured to analyze the liquid protein sample pumped out of the heated tube.

[0068] Computer software is also provided. The computer software comprises instructions which, when executed on a processor, cause the processor to perform the method as described above.

[0069] Other examples useful for understanding the present invention are listed in the following numbered clauses:

[0070] Clause 1. A method for detecting a target protein of one or more compounds added to an unpurified complex protein mixture, the method comprising:

[0071] (a) preparing a liquid sample consisting of a protein mixture or living cells with or without one or more test compounds;

[0072] (b) filling a tube maintained at a fixed temperature completely or partially with the sample;

[0073] (c) eluting the sample from the tube after a time period ranging from zero minutes to several minutes.

[0074] Clause 2. The method of clause 1, wherein the elution rate of the sample is made significantly different from the filling rate such that different volumes of the sample are exposed to the temperature for different time intervals.

[0075] Clause 3. The method of clause 1, wherein the filling rate or the elution rate or both rates are varied during filling or elution such that different volumes of the sample are exposed to the temperature for different time intervals.

[0076] Item 4. A device utilizing the method according to Item 1, comprising at least the following modules: an autosampler, a pump, a tube maintained at a constant temperature and a fraction collector, wherein the autosampler contains the protein sample and the pump enables the tube to be filled with the protein sample and they are eluted from the tube to the fraction collector.

[0077] Clause 5. The apparatus according to clause 4, wherein all mentioned modules are programmable in software or user interface, and all operation steps are programmed in the software or user interface before running the sample.

[0078] Additionally, apparatus for automating the methods of the present invention are described. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 The thermal proteome profiling (TPP) method is shown.

[0080] Figure 2 The Proteomic Integrated Solubility Alteration (PISA) method is shown.

[0081] Figure 3 A shows an example device and the flow of a liquid protein sample through the example device.

[0082] Figure 3 B shows Figure 3 A is a graph of the flow rate of a liquid protein sample versus time for the apparatus shown.

[0083] Figure 4 shows the incubation time of a portion of the sample relative to Figure 3 The position of the sample in the tube of the device A and Figure 3 Graph of the flow rate of B.

[0084] Figure 5 A shows another example device.

[0085] Figure 5 B shows Figure 5 A is a graph of the flow rate of a liquid protein sample versus time for the apparatus shown.

[0086] Figure 6Results from a specific example approach are shown. DETAILED DESCRIPTION

[0087] One approach to understanding how one or more active molecules (e.g., drugs) affect each of the different proteins within a proteome is "thermal proteome profiling" (TPP), which relies on changes in protein thermal stability to assess how molecules affect different proteins within a proteome. As described by Savitski et al., this involves:

[0088] Divide the sample into several fractions.

[0089] heating each fraction to a different temperature (in some examples, using 10 different fractions for each of 10 different temperatures), and

[0090] A "melting curve" is determined for each protein in the sample.

[0091] The melting curve is a plot of protein abundance versus temperature (see Figure 1 ).

[0092] Typically, the melting curve of a "target" protein bound to a molecule is shifted to higher temperatures compared to the melting curve of a protein not bound to the molecule. This may be due, for example, to an increase in the thermal stability of the protein when bound to a molecule.

[0093] A related approach is the "Proteome-wide Solubility Alteration" (PISA) assay, examples of which are described in Gaetani et al. and Sabatier et al., providing an alternative to TPP in which samples can be analyzed together. This may therefore improve the throughput of proteome analysis methods.

[0094] Some example methods enable the analysis of changes in protein three-dimensional structure by providing differential solubility assays, in which two or more proteome states are compared in terms of the solubility differences of individual proteins in these states. Since most proteins exist in cells in soluble and insoluble states (Vecchi et al., PNAS 2020, 117, 1015-1020), almost all known proteomics protocols include steps for separating the soluble and insoluble parts of a proteome. Most proteomics methods are based on measuring the abundance of soluble proteins as a measure of protein concentration in a sample. Soluble protein abundance can be used as a measure of protein solubility in a given proteome state.

[0095] To ensure that not all molecules of a given protein are fully soluble in any proteome state, and thus increase the dynamic range of the solubility measurement, part of the sample is incubated at a different temperature compared to ambient temperature. Many proteins become less soluble at higher temperatures, but some lose their solubility at temperatures close to the freezing point. Furthermore, if related proteins are from different organisms, they may have different solubilities. Therefore, the optimal temperature for incubation will depend on the specifics of the protein of interest.

[0096] The present invention describes a method and apparatus for commercial automation of TPP or PISA methods. The original TPP and PISA methods involved dividing the sample into many (e.g., about 10) fractions and incubating each fraction at different temperatures. This is a relatively complex, time-consuming manual task and uses a relatively large amount of sample (e.g., about 10×about 10 μL).

[0097] In the prior art TPP and PISA methods, incubation is performed at different temperatures but for the same time. In contrast, the present invention obtains the same (or similar) information using only one amount of sample ("one pot" operation) in a significantly less complex and suitable for automation.

[0098] In some examples, the method relies on different incubation times rather than multiple different temperatures. Different portions of the liquid protein sample are incubated for different times. In such examples, the incubation temperature can be a fixed temperature.

[0099] To achieve this, the volume in which the incubation takes place is spatially extended, such as in a tube or capillary, and different parts of the sample are exposed to a fixed temperature for different periods of time. Figure 3 A shows an example apparatus and flow of a liquid protein sample through a heated tube implementing some example methods. Figure 3 B shows a graph of flow rate of a liquid protein sample versus time for some example methods. Figure 4 Shown is a graph of the incubation time of a portion of a sample versus the position of the sample in the tube.

[0100] Figure 5 Another example device is shown in A. Figure 5 B shows a graph of flow rate of a liquid protein sample versus time for other example methods.

[0101] In a first example, the sample is loaded into a long capillary tube, which is heated at a fixed temperature, such as by being attached to a heating plate ("hot coil"). The speed at which the sample is loaded into and removed (eluted) from the tube can be controlled individually by one or more pumps. The sample is loaded into the heated tube over a relatively short period of time (e.g., over a period of about 10 seconds). The sample is then eluted from the heated tube over a relatively long period of time (e.g., over a period of more than about 10 minutes). Those portions of the sample that are eluted from the heated tube relatively early in the experiment will be incubated for a shorter amount of time, while those portions of the sample that are eluted from the heated tube relatively late in the experiment will be incubated for a longer amount of time. Then, by analyzing different portions of the sample that have been subjected to different incubation times, a "melting curve" for each protein in the sample can be obtained.

[0102] It has been shown that the method can be automated and works well with small amounts of sample (eg, about 0.9 μg).

[0103] In a second example, a long capillary can be attached to a heating plate with two (or more) heaters maintained at different temperatures so that a temperature gradient is maintained across the plate / capillary. All samples can be retained in the capillary for the same period of time. Due to the temperature gradient, different parts of the sample within the capillary will experience different temperatures.

[0104] Although mass spectrometry is preferably used to measure the abundance of each protein, in principle other techniques such as Western blotting, mass spectrometry, etc. may also be used or used instead.

[0105] A known problem with this method is cross contamination of the sample (due to the sample sticking to the tube walls). This can be solved by washing and / or passivating the tube before each measurement.

[0106] Two heating plates may be used instead of a single heating plate, for example with the tube clamped between the two heating plates. Alternatively, a heated wire may be wound around the tube.

[0107] In one specific example, an apparatus includes:

[0108] A) An autosampler comprising an input port for a liquid sample with a protein mixture or living cells and a needle capable of collecting a defined volume of said liquid sample. The autosampler may have a cooling system that keeps the sample stable by cooling it before processing in the rest of the device.

[0109] B) A pump capable of transferring the volume from the needle into a tube maintained at a constant temperature, for example by attaching it to a tube made of a material with a high heat capacity (such as metal or ceramic)

[0110] Advantageously, the tube is also made of a material with a high heat capacity, such as fused silica.

[0111] C) An electronic temperature control system capable of maintaining a fixed temperature of the plate elevated compared to the ambient temperature.

[0112] D) A fraction collector capable of receiving liquid sample volumes eluted through the tube and collecting them into output wells.

[0113] E) Software program that operates the needle, pump, control system and fraction collector.

[0114] A specific example method includes the following steps:

[0115] a) Preparation step: Prepare a liquid sample containing cell lysate or intact cells with or without a molecule of interest (such as a drug), and deposit the prepared sample into a given input well of the autosampler.

[0116] b) Processing step: using a pump, the liquid sample is transferred from a given input hole into a tube, and then the liquid sample is eluted from the tube into a fraction collector, so that different volumes of sample are exposed to the temperature at which the tube is maintained for different periods of time. This can be achieved by filling the tube at a different constant rate compared to the constant rate of elution from the tube, or by using a variable rate of filling the tube, or by using a variable rate of eluting the sample from the tube, or by a combination thereof.

[0117] c) Collection step: Using a fraction collector, the eluted samples are deposited into the output wells corresponding to the input wells from which a given sample originated.

[0118] d) Lysis step (only for samples containing intact cells): the intact cells are lysed and a cell lysate is obtained.

[0119] e) Centrifugation step: Each sample lysate was centrifuged to separate soluble proteins from insoluble proteins.

[0120] f) Analytical step: The soluble proteins obtained in the centrifugation step are analyzed by quantitative proteomics.

[0121] g) Data processing step: Compare protein relative abundance and identify proteins whose abundance, and therefore solubility, changes in the presence of drug.

[0122] Some example methods can be used to analyze single proteins and protein mixtures, including proteins from unfractionated cell lysates and intact cells. Other example methods can be used to assess changes in protein solubility due to thermal unfolding. One area of ​​application is to identify proteins in unpurified complex samples that interact with a test compound of interest added to the mixture.

[0123] In the simplest case of two proteome states, the readout of the differential solubility assay is the difference or ratio of abundance between the two states for each protein. Such measurements performed in many replicates provide statistical significance of the difference or ratio by a suitable statistical test, such as a Student's t-test.

[0124] Figure 6 Results from a specific example are shown. Cell lysates were treated with methotrexate (MTX) and controls were treated with vehicle (DMSO). Five replicates of 5 μL samples were placed in an autosampler. The autoinjector loaded 3 μL samples into the OPTI-PISA capillary one by one at high speed (<1 min). After a short time, the sample was slowly eluted from the OPTI-PISA capillary into the fraction collector (≥10 min). The collected samples were digested, TMT labeled and analyzed by LC-MS / MS. The MTX target DHFR was identified as an outlier in the volcano plot with an excellent p-value (<10 -8 ). Only 900 ng of lysate was used per sample. The results showed better p-values ​​than in the equivalent manual PISA experiment. This improvement can be attributed at least in part to the automation of sample incubation.

[0125] Therefore, described OPTI-PISA method provides many advantages.Some methods can make the heat treatment of PISA experiment of lysate fully automated.In some embodiments, sample consumption is low compared with prior art methods, lower than 1 μ g / sample in some examples.Therefore, due to reliable automation, some example methods provide the high reproducibility of result.Therefore, the result can provide better (lower) p value than in the manual PISA experiment on the same sample.Some example methods can cause the processing time of about 10 minutes-15 minutes for each sample.Therefore, compared with prior art methods, described method provides high throughput.Described method also can be compatible with automated proteome analysis equipment and software.

[0126] In the description of the present invention herein, it should be understood that, unless otherwise implicitly or explicitly understood or stated, words appearing in the singular encompass their plural counterparts, and words appearing in the plural encompass their singular counterparts. In addition, it should be understood that, unless otherwise implicitly or explicitly understood or stated, for any given component or embodiment described herein, any possible candidate or alternative listed for the component may generally be used individually or in combination with each other. In addition, it should be understood that the drawings as shown herein may not be drawn to scale, wherein only some elements may be drawn for the clarity of the present invention. And, reference numerals may be repeated in each of the drawings to show corresponding or similar elements. In addition, it should be understood that, unless otherwise implicitly or explicitly understood or stated, any list of such candidate or alternative is only exemplary and non-restrictive.

[0127] Unless otherwise defined, all other technical terms and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. In the event of a conflict, this specification (including definitions) shall prevail. It should be understood that there is an implied "about" before the quantitative terms mentioned in this specification, so that slight and insubstantial deviations are within the scope of this teaching. In this application, unless otherwise expressly provided, the use of the singular includes the plural. In addition, the use of "comprising", "containing" and "including" is not intended to be restrictive. As used herein, "one" or "one" may also refer to "at least one" or "one or more". In addition, the use of "or" is inclusive, so that when "A" is true, "B" is true or "A" and "B" are both true, the phrase "A or B" is true.

Claims

1. A method for preparing a liquid protein sample for proteomic analysis, the method comprising: pumping a liquid protein sample into a heated tube, wherein the liquid protein sample comprises a plurality of portions; incubating the liquid protein sample in the heated tube, wherein the incubation conditions for a first portion of the sample are different from the incubation conditions for a second portion of the sample; The liquid protein sample is pumped out of the heated tube.

2. The method of claim 1, wherein the incubation conditions include a time period for incubating the corresponding portion of the sample in the heated tube.

3. The method according to claim 1 or claim 2, wherein the heating tube is heated to a predetermined temperature.

4. A method according to any preceding claim, wherein each of the plurality of portions is incubated in the heating tube for a different period of time.

5. A method according to any preceding claim, wherein a fill rate, which is the pumping rate used to pump the liquid protein sample into the heated tube, is significantly different from an elution rate, which is the pumping rate used to pump the liquid protein sample out of the heated tube.

6. The method of claim 5, wherein the filling speed and / or the elution speed is varied during filling and / or elution such that each of the plurality of portions is incubated in the heating tube for a different period of time.

7. A method according to any preceding claim, wherein incubating the sample in the tube comprises pumping the sample through the heated tube at a controlled rate such that an incubation period is defined as the time it takes for the corresponding portion of the liquid protein sample to travel from the inlet of the heated tube to the outlet of the heated tube.

8. A method according to any preceding claim, wherein each of the plurality of portions is incubated in the heating tube for a different period of time on average, such that the average incubation time of a first portion of the sample is different from the average incubation time of a second portion of the sample.

9. The method of claim 1, wherein the incubation conditions include the temperature at which the corresponding portion of the sample is incubated in the heating tube.

10. The method of claim 9, wherein the heating tube is heated to a temperature gradient or a plurality of different temperatures.

11. The method of claim 9 or claim 10, wherein incubating the sample in the heated tube comprises keeping 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.

12. The method according to any one of claims 9 to 11, wherein the first part and the second part are incubated in the heating tube at average different temperatures.

13. A method according to any preceding claim, wherein the incubation conditions are different for each of the plurality of portions.

14. The method of any preceding claim, further comprising pumping the liquid protein sample into an unheated tube in fluid communication with the heated tube.

15. The method of claim 14, wherein pumping the liquid protein sample into the heated tube and / or pumping the liquid protein sample out of the heated tube comprises pumping a wash solvent into the unheated tube.

16. The method of claim 15, further comprising generating bubbles in the unheated tube between the liquid protein sample and the wash solvent.

17. A method according to any preceding claim, further comprising cooling the liquid protein sample after it has been pumped out of the heated tube.

18. The method of any preceding claim, further comprising analyzing the liquid protein sample to determine the relative abundance of one or more proteins in the sample.

19. The method of any one of claims 1 to 17, wherein pumping the liquid protein sample out of the heated tube comprises pumping the liquid protein sample out of the heated tube in a plurality of fractions.

20. The method of claim 19, further comprising analyzing each fraction of the liquid protein sample individually to determine the relative abundance of one or more proteins in each fraction.

21. The method according to any preceding claim, wherein the liquid protein sample comprises a protein mixture and / or living cells.

22. 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.

23. An apparatus configured to perform the method according to any one of claims 1 to 22, the apparatus comprising: Heating tube, A pump is configured to pump the liquid protein sample into the heated tube and pump the liquid protein sample out of the heated tube.

24. The apparatus of claim 23, further comprising one or more of the following: a fraction collector configured to collect the liquid protein sample pumped out of the heated tube, and An analytical instrument is configured to analyze the liquid protein sample pumped out of the heated tube.

25. 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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