Seeded precipitation of polypeptides

Abstract

The present invention relates to a method for processing or fractionating a sample containing proteins, polypeptides and / or peptides, comprising: (a) altering the physicochemical conditions of the sample; and (b) performing one or both of the following steps (i) and (ii): (i) adding solid particulate matter to the sample; and (ii) performing the method in a vessel having a rough surface, wherein steps (a) and (b) can be performed simultaneously or in any order; and wherein the processing or fractionation obtains one or more first fractions of proteins, polypeptides and / or peptides as a precipitate on the particulate matter and / or on the heterogeneous surface, and a second fraction of proteins, polypeptides and / or peptides remaining in the supernatant.

Description

Detailed Description of the Invention

[0001] The present invention relates to a method for processing or fractionating a sample comprising proteins, polypeptides and / or peptides, comprising the steps of: (a) altering the physicochemical conditions of said sample; and (b) performing one or both of the following steps (i) and (ii): (i) adding solid particulate matter to said sample; and (ii) performing said method in a vessel having a rough surface, wherein steps (a) and (b) can be performed simultaneously or in any order; said processing or fractionation obtaining one or more first fractions of proteins, polypeptides and / or peptides as a precipitate on said particulate matter and / or said heterogeneous surface, and a second fraction of proteins, polypeptides and / or peptides remaining in the supernatant.

[0002] In this specification, several documents are cited, including patent applications and manufacturer's manuals.The disclosures of these documents are not considered relevant to the patentability of this invention, but are incorporated herein by reference in their entirety.More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0003] Fractionation, separation, or depletion of polypeptides / proteins remains a challenge in biopharmaceutical research. For example, one of the major goals in current pharmaceutical research is the identification, detection, and quantification of polypeptide / protein biomarkers in bodily fluids such as plasma. Due to the very high dynamic range of polypeptides / proteins in bodily fluids, low abundance polypeptides / proteins are often difficult or often impossible to detect. To this end, many current methods aim to deplete and remove high abundance polypeptides / proteins, such as serum albumins and immunoglobulins, by selective binding to proteins with higher affinity for the respective proteins. For example, protein G beads are commonly used to reduce the amount of IgG proteins in a sample of interest. In addition, fractionation methods such as ion exchange chromatography are also often used to separate polypeptides / proteins and reduce the complexity and dynamic range of the sample.

[0004] The concept of selective protein / polypeptide precipitation is well established and has been described previously (EJ Cohn, LE Strong, WL Hughes, DJ Mulford, JN Ashworth, M. Melin, and HL Taylor; Journal of the American Chemical Society, 1946, Vol. 68(3), pp. 459-475; DOI:10.1021 / ja01207a034). Here, pH, temperature, and ethanol content are utilized to induce stepwise precipitation of certain polypeptides / proteins in solution in plasma. This process is still one of the major processes in large-scale protein enrichment in the pharmaceutical industry to purify certain protein / polypeptide species. However, this process requires large amounts of starting material to generate sufficient precipitate that can be filtered or spun down to successfully separate the precipitated polypeptides / proteins from the remaining soluble polypeptides / proteins. Furthermore, automation of filtration or centrifugation remains difficult.

[0005] Batth et al. (Molecular and Cellular Proteomics 18, 1027-1035, 2019) and Hughes et al. (Molecular Systems Biology 10, 757, 2014) describe workflows for proteomic analysis that share harsh conditions involving protein denaturation, e.g., temperatures of 80°C even at boiling, high concentrations of denaturing detergents, e.g., SDS, high concentrations of organic solvents, e.g., acetonitrile, or combinations thereof.

[0006] Considering the shortcomings of the prior art, the technical problem underlying the present invention can be seen in improved means and methods for sample processing or sample fractionation, in particular in the field of proteomics, including the provision of samples comprising polypeptides, or in research, production and diagnostics.

[0007] This technical problem has been solved as evidenced by the enclosed claims and by the examples contained in this disclosure.

[0008] The present invention therefore relates in a first aspect to a method for processing or fractionating a sample comprising proteins, polypeptides and / or peptides, comprising the steps of: (a) altering the physicochemical conditions of said sample; and (b) performing one or both of the following steps (i) and (ii): (i) adding solid particulate matter to said sample; and (ii) carrying out said method in a vessel having a rough surface, wherein steps (a) and (b) can be carried out simultaneously or in any order; said processing or fractionation obtaining one or more first fractions of proteins, polypeptides and / or peptides as a precipitate on said particulate matter and / or said heterogeneous surface, and a second fraction of proteins, polypeptides and / or peptides remaining in the supernatant.

[0009] In connection therewith, the present invention provides a method for processing or fractionating a sample containing biomolecules, comprising: (a) altering the physicochemical conditions of the sample; and (b) performing one or both of the following steps (i) and (ii): (i) adding solid particulate matter to the sample; and (ii) performing the method in a vessel having a rough surface, wherein steps (a) and (b) can be performed simultaneously or in any order.

[0010] Preferred biomolecules are biopolymers. Preferred biopolymers include nucleic acids, sugars and lipids in addition to the proteins, polypeptides and peptides disclosed above.

[0011] The term "fractionation" has its industry-established meaning. It refers to the division of a mixture of compounds or analytes into at least two separate mixtures, where the separate mixtures differ in their relative content with respect to at least one of the compounds or analytes. In other words, at least one analyte is enriched in at least one fraction compared to at least one other fraction. Fractionation may, but need not, result in a substantial or total depletion of at least one analyte from at least one fraction. In a more general sense, fractionation may result in an alteration or reduction in dynamic range or complexity compared to the original mixture, for example, the ratio of the amount of the most abundant analyte to the amount of the least abundant analyte may be reduced in at least one of the resulting fractions compared to the original mixture. Such a reduction in dynamic range generally increases the amount of analytes that are suitable for detection in a subsequent analytical procedure, for example mass spectrometry (MS). As used herein, the term "complexity" refers to the number of analytes, particularly proteins, polypeptides and / or peptides, contained in a given fraction. Compression of the dynamic range generally involves detecting a greater number of analytes.

[0012] The terms "protein", "polypeptide" and "peptide" likewise have their established meaning in the art. In particular, peptides and polypeptides are polycondensates of amino acids, preferably L-alpha-amino acids that constitute proteins. In the present specification, such polycondensates are referred to as peptides if they contain 30 amino acids or less, and longer polycondensates are referred to as polypeptides. Preferred peptides are those having a length of 20-30 amino acids. Generally, peptides and polypeptides are single chains, not excluding the presence of cross-links such as disulfides and / or modifications such as phosphorylation. On the other hand, proteins may have a more complex structure in that they may be oligomers of polypeptides and / or contain non-proteinaceous components such as prosthetic groups. The analysis of such proteins, polypeptides and peptides is of particular interest with regard to distinguishing between healthy and disease states at the molecular level.

[0013] "Physicochemical conditions" encompass both physical parameters, such as temperature, as well as chemical parameters, such as solvent composition. Physicochemical conditions have an effect on the state of the analytes in the mixture, which can be expressed in terms of the chemical potential of the analytes. A change in physicochemical conditions is a means to induce, to a greater or lesser extent, the precipitation of one or more analytes in the mixture. The change in conditions promotes the precipitation state of the analytes. In this respect, a line must be drawn between precipitation and denaturation. As will become clearer below, preference is given to conditions that induce precipitation but avoid denaturation.

[0014] The term "precipitation", especially when used in the field of chemistry, has its established meaning in the industry. It refers to the change in state of the composition of matter under consideration (here proteins, polypeptides and / or peptides) from dissolved or soluble to insoluble. The insoluble material may initially form as a suspension and then settle, for example under the influence of gravity (which can be facilitated by procedures such as centrifugation) to obtain a sediment (insoluble material in a specific area, typically at the bottom of the vessel or container used). In the case of centrifugation, the sediment is typically referred to as a pellet.

[0015] Said processing or fractionation obtains one or more first fractions of proteins, polypeptides and / or peptides as sediments on said particulate matter and / or said heterogeneous surface, and a second fraction of proteins, polypeptides and / or peptides remaining in the supernatant. Such fractionation is in principle inherent to the method of the first embodiment in its broadest definition. This raises the terms supernatant and sediment, both of which have their industry-established meanings. After processing according to the method of the first embodiment, a portion of the proteins, polypeptides and peptides of the original sample have changed their state of matter and are no longer in solution, but adhere to said heterogeneous surface and / or said solid particulate matter as sediments. The remaining analytes are still in solution and constitute the supernatant.

[0016] This raises the concept of at least one first fraction and a second fraction. The fact that there may be more than one first fraction, i.e. more than one sediment, stems from the fact that the method of the invention may use more than one type of particulate matter and / or more than one type of heterogeneous surface. One type of particulate matter or heterogeneous surface is preferred.

[0017] Sedimentation occurs when a solution becomes supersaturated, i.e., when the solution contains more solute than can be stably maintained in the solution under given conditions. However, the supersaturated state may persist for a certain amount of time. This is why sedimentation occurs, and generally, a process known as nucleation must occur. If nucleation does not occur, the supersaturated state may become metastable. "Metastable" refers to a state that is stable in response to a small change in conditions, but unstable when a larger change in conditions occurs. Nucleation may be facilitated by seeding. Seeding generally converts a metastable supersaturated state into an unstable state with sedimentation. Seeding is an important feature of the present invention in mechanistic terms, as will become clearer below.

[0018] Physicochemical conditions have been described as suitable inducers of precipitation; see the references cited in the background section above. A list of preferred physicochemical conditions to be varied according to the invention is the subject of preferred embodiments further disclosed below. The term "harsh" in relation to conditions as used herein refers to physicochemical conditions that are particularly suitable for inducing precipitation. Harsh conditions may, in extreme cases, cause the precipitation of all or substantially all proteins, polypeptides and / or peptides from said sample. Therefore, harsh conditions are generally less preferred. Another drawback of harsh conditions is that, in addition to precipitation, they may induce denaturation of biomolecules such as proteins, polypeptides and peptides, which are preferred analytes. As will be considered in more detail below, the means (b) according to the invention allows the use of less harsh conditions, including conditions that do not induce precipitation at all or to an insufficient extent and therefore cannot be used in the absence of the means (b). Preferred less harsh conditions are non-denaturing conditions as well as specific conditions further disclosed below.

[0019] Indeed, the inventors have surprisingly discovered that the effect of the change in physicochemical conditions can be modulated or enhanced. This is expressed in step (b) of the method of the first aspect of the invention. Such modulation or enhancement may, but need not, affect different analytes to different degrees. The measures according to step (b) are physical in nature. Their overarching principle is a structured or rough surface. Such structured surfaces can be embodied as particulate matter added to the sample to be processed or fractionated, or as a modification of the surface of the container in which the processing or fractionation takes place.

[0020] The structured surface is not particularly limited. Surprisingly, it has been found that the composition of the particulate matter is irrelevant. Furthermore, the structured surface of the container may be made of the same material (glass, plastic, etc.) as the rest of the container.

[0021] The surface of the container may be rough overall or in one or more regions. The size of said one or more regions is macroscopic, while the structural non-uniformity or roughness in the rough region is microscopic. For example, the region may have a diameter of 1 to 20 mm, such as 2 to 10 mm, including 3, 4, 5, 6, 7, 8, or 9 mm. On the other hand, the structural non-uniformity or roughness is on the nanometer to μm scale, preferably 0.4 to 100 μm, more preferably 1 to 60 μm, such as 2 to 30 μm, including 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 and 25 μm.

[0022] The structural inhomogeneity or roughness of a surface can be described in terms of the roughness parameter Ra. Ra is the arithmetic mean deviation from the centerline of the profile along the surface under consideration. The value of Ra is a length; it is usually specified in μm. In a preferred embodiment, the preferred values ​​given above for roughness are the Ra values. Roughness can be determined by instruments such as profilometers, optical comparators, interferometers and microscopes. A preferred instrument is a profilometer.

[0023] Generally, the preferred scale of the structural heterogeneity is also the preferred size of said solid particulate matter.

[0024] Without wishing to be bound by any particular theory, it is believed that the solid particulate matter and the rough surface stabilize very small nuclei (initial intermediates in the precipitation process) that always form but dissolve again in the absence of particles or rough surfaces. To explain further, small nuclei have a larger surface to volume ratio compared to larger nuclei. The free energy of a nucleus depends both on its surface and its volume. According to classical nucleation theory, the surface term is always positive and does not favor nucleation such that the total free energy of nucleation is positive for small nuclei. Only when the size of the nuclei is growing, the volume term dominates and finally, nucleation proceeds and precipitation occurs. It is believed that the particulate matter or the rough surface modifies the surface energy of small nuclei such that the dominance of the volume term already occurs for very small nuclei.

[0025] The method of the present invention performs better when compared with established methods in terms of reducing the dynamic range of analytes in a sample and / or increasing the number of analytes suitable for detection.The reduction of dynamic range improves the performance of any downstream analytical method, such as mass spectrometry.Analysis methods, such as mass spectrometry, are essentially characterized by a limited dynamic range within which sufficient qualitative and quantitative detection is possible.By reducing the dynamic range of the analytes contained in a sample, for example by reducing the relative amount of the most abundant analyte, low abundance analytes that escape detection in samples that are not processed using the method of the present invention become detectable and quantifiable.

[0026] The methods of the invention further provide a dramatic improvement in processing speed compared to industry-established methods of protein or polypeptide fractionation: while overnight or days of processing are common in the art and are also contemplated by the invention (any processing time from 30 seconds to overnight (about 12 hours) or one week is preferred), the methods of the invention can more preferably be carried out on a time scale measured in minutes, e.g., 1 minute to 3 hours, 2 to 30 minutes, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 minutes.

[0027] A further improvement is the amount of sample sufficient for successful implementation of the method. Whereas prior art methods generally require large amounts, or even liters, of body fluid, the present invention works with small sample sizes, such as 1-1000 μl, 5-100 μl, e.g., 10, 20, 30, 40 or 50 μl. For body fluids with low protein / polypeptide / peptide concentrations, such as urine, larger sample sizes are also contemplated, such as 1 μl-100 ml or 10 μl-50 ml, in addition to the ranges and values ​​specified above.

[0028] As will be seen in the examples, the proteome under consideration could be analyzed at an unprecedented depth, i.e. the number of proteins amenable to detection by MS was unexpectedly high.

[0029] A further advantage of the present invention, particularly the modulating / enhancing effect on the sedimentation of heterogeneous surfaces or particulate matter, is that physicochemical conditions can be used in a less harsh manner compared to procedures established in the industry. In other words, in the presence of the measure according to step (b) of the first aspect, the physicochemical conditions can be changed to a lesser extent compared to the absence of said measure, while still obtaining satisfactory results in terms of both sedimentation and fractionation. In fact, it has been found that less strongly sedimenting conditions are not only sufficient but also better for separating low abundance from high abundance proteins, polypeptides or peptides, for example in plasma samples. While strongly sedimenting conditions (such as those described in Cohn et al., supra) generally result in less selective sedimentation of mixtures of proteins, polypeptides and / or peptides, it has been surprisingly found that less strongly sedimenting conditions result in more selective sedimentation of seeded sediments. As shown in the examples, it was possible to enrich low abundance analytes compared to high abundance analytes in the sediments. In particular, it is envisaged or even preferred to precipitate only a fraction of the total proteins, polypeptides and / or peptides in said sample. By doing so, the dynamic range and the number of detectable proteins, polypeptides and / or peptides are modified in a preferred manner. Said fraction may be in the range of 0.01-50% by weight, more preferably 0.1-20% by weight, for example 0.5-10% by weight. Even smaller fractions, such as 0.0001-5% by weight and 0.001-1% by weight, are purposefully envisaged.

[0030] As shown in the Examples, because the methods of the invention allow for the detection of a greater number of proteins, polypeptides and / or peptides in a given sample (in other words, the proteome is detected at greater depth), the methods provide novel markers and combinations of novel markers for a given state ("state" including health and disease) of a cell, tissue or organism, which in turn provide improved diagnostics. Proteins are particularly preferred analytes, especially considering that detection of proteins is of interest in many analytical or diagnostic applications.

[0031] In a preferred embodiment, said proteins, polypeptides and / or peptides are dissolved in said sample.

[0032] In a further preferred embodiment, said sample is a cell lysate or a body fluid, optionally said body fluid or cell lysate being treated with an anticoagulant.

[0033] The application of the method of the present invention to cell lysates extends to what is known in the art as "host cell protein (HCP)" analysis. HCP analysis refers to the monitoring of the purification of a biopharmaceutical, e.g., an antibody, from the cells, e.g., immortalized B cells, used for its production / manufacturing. Such purification is generally a stepwise process of depleting host cell proteins, ultimately obtaining a pharmaceutical grade product. Said host cell proteins include potentially allergenic or otherwise undesirable proteins or polypeptides derived from host cells. In other words, HCP analysis can be performed using the method of the present invention.

[0034] Body fluids include blood, serum, plasma, saliva, nasal swabs, urine, vaginal fluid, semen, and cerebrospinal fluid. Cell lysates can be obtained from any cell, tissue, or biopsy. Preferably, the insoluble portion of the cell lysate is removed, for example by centrifugation or filtration, before carrying out the method of the present invention.

[0035] The term "antibody" has its art-established meaning. It encompasses all naturally occurring classes and subclasses, such as IgG (including IgG1, IgG2, IgG3 and IgG4), IgA, IgM, IgD and IgE, as well as camelid antibodies. As used herein, the term "antibody" further encompasses fragments (such as Fab) and engineered antigen-binding molecules, such as single chain antibodies, bi- or multispecific antibodies, and any antigen-binding molecule having at least three (particularly in the case of camelid antibodies and their derivatives), preferably six, complementarity determining regions (CDRs).

[0036] The treatment of biological samples with anticoagulants is common in the art, especially when it becomes a clinical sample. For example, serum is the liquid component of blood that can clot when stored, but the addition of an anticoagulant helps prevent clotting, resulting in plasma. Blood clots in the case of serum and blood cells in the case of plasma are generally removed by centrifugation. Anticoagulants are known in the art, with EDTA being particularly common, and EDTA K2 and EDTA K3 being preferred.

[0037] In a further preferred embodiment, step (a), step (b), or both steps (a) and (b) are preferably carried out more than once with the supernatant defined above.The method of the present invention can be carried out repeatedly.For example, once the first sediment has been removed, the method can be repeated with the supernatant under the same or modified physicochemical conditions and / or with the same or different heterogeneous surface or particulate matter.This allows the supernatant obtained during the first implementation of the method to be further fractionated.

[0038] In a further preferred embodiment, said physicochemical conditions during said change do not include (i) a temperature above 70° C., a concentration of denaturing detergents such as SDS above 0.8% (w / v), and a concentration of organic solvents such as acetonitrile above 25% (v / v); and / or (ii) are non-denaturing conditions for said proteins, polypeptides and / or peptides.

[0039] Commonly used workflows for sample preparation (see the introductory part of this specification) involve the complete or substantially complete precipitation of proteins, polypeptides and peptides. For this purpose, very harsh conditions are used, in particular those described in item (i) of the above embodiment. On the other hand, the method of the present invention, thanks to the use of particles and / or rough surfaces, allows to dispense with such harsh conditions.

[0040] This offers a clear advantage. As mentioned above, the proteome is generally characterized by a large dynamic range. As a result, the detection of low abundance proteins in the presence of high abundance proteins such as antibodies and albumin becomes difficult. However, for many applications in the fields of research and diagnostics, the capture of low abundance proteins is interesting. For example, in disease states, low abundance markers characteristic of the disease leave the affected local tissue and enter the plasma, where they are highly diluted. Compression of the dynamic range is therefore desirable. The use of particulate matter and / or rough surfaces is one means to achieve this. The avoidance of harsh conditions, in particular denaturing conditions, is a further means. To further explain, comprehensive denaturation and simultaneous precipitation generally leaves the dynamic range substantially unchanged: all analytes are precipitated and subsequently returned to solution for further processing. On the other hand, less harsh conditions (for their preferred implementation see below) have a more differentiated effect. In particular, experience has shown that high abundance proteins described are less likely to sediment under non-denaturing conditions than low abundance proteins described (although they will do so under the conditions previously outlined herein). As a result, precipitation under non-denaturing conditions preferentially, but generally not completely, depletes the high abundance proteins from the precipitate. Thus, what is found in the precipitate, while still highly informative, is characterized by a compressed dynamic range.

[0041] Therefore, preferred conditions according to the present invention are non-denaturing conditions. As used herein, the term "conditions", unless otherwise specified, refers to the conditions after "changing the physicochemical conditions" according to step (a) of the method of the first aspect. Assuming that said sample comprising proteins, polypeptides and / or peptides comprises said proteins, polypeptides and / or peptides in their native state, i.e. in a non-denatured state, it is understood that said conditions prior to said change are generally non-denaturing, e.g. because said sample was obtained from its native biological environment without or substantially without changing its physicochemical conditions, at least not to a denaturing extent.

[0042] The term "non-denaturing conditions" for proteins, polypeptides and peptides is commonly used in the art and understood by those skilled in the art. The process of denaturation results in the loss of defined three-dimensional structure. Such loss generally impairs "function", including the ability of an enzyme to bind to its cognate binding partner and / or process its cognate substrate. Moreover, said loss is generally accompanied by an increase in size (e.g., measured in units of hydrodynamic radius) and an increase in the width of the size distribution.

[0043] For completeness, a preferred definition of the threshold that distinguishes between non-denaturing and denaturing conditions is as follows:

[0044] Dynamic light scattering (DLS) is a method to calculate the diffusion constant D in terms of the hydrodynamic radius R h The hydrodynamic radius R of the particle is given by the Stroke-Einstein relation h It is an analytical method that can be used to determine the size distribution of small particles in a suspension or solution by measuring the size distribution of the molecules in the suspension or solution. It allows to distinguish between the non-denatured and the denatured states of proteins, polypeptides and peptides. The non-denatured or native state is characterized by a well-defined structure that, as mentioned above, gives a peak in the size distribution with a limited width. Denaturation, on the other hand, causes the loss of said well-defined structure, and the previously homogeneous population of molecules that has all or substantially all of said well-defined structure shares the lack of the native structure, but otherwise, in particular its hydrodynamic radius R hThe native Rh is transformed into a more heterogeneous collection of disordered structures that are diverse in terms of Rh. This is reflected by the broadening of the peak of the distribution obtained by DLS, which is generally accompanied by an increase in the mean hydrodynamic radius. An increase in the mean Rh as well as a broadening of the peak of the distribution of Rh, preferably a statistically significant change in either parameter, are both hallmarks of denaturation. The threshold value for distinguishing native from denatured according to the present invention takes full advantage of the change in peak width.

[0045] A model-independent measure of the width of a peak is the full width at half maximum (FWHM). More specifically, the FWHM is the difference between two values ​​of the independent variable where the dependent variable is equal to half its maximum value. In the case of a Gaussian distribution that defines the shape of the peak, the FWHM is a simple function of the standard deviation of the Gaussian distribution.

[0046] For the purpose of defining the threshold value that distinguishes between denaturing and non-denaturing conditions, in principle any protein can be used. Preferred proteins in this regard are preferably GAPDH and MTAP of human origin, more preferably human GAPDH.

[0047] Preferred non-denaturing conditions are physiological conditions. The term "physiological conditions" has its art-established meaning and refers to intracellular conditions. Preferred physiological conditions are as follows: 14 mM Na + , 140 mM K + , 10 -7 mM Ca 2+ , 20mM Mg 2+ , 4 mM Cl - , 10mM HCO3 - , 11mM HPO4 2- and H2PO4 - , 1mM SO4 2- , 45 mM phosphocreatine, 14 mM carnosine, 8 mM amino acids, 9 mM creatine, 1.5 mM lactate, 5 mM ATP, 3.7 mM hexose monophosphate, 4 mM protein and 4 mM urea. The preferred temperature is 37° C. The preferred pressure is 1013.25 hPa.

[0048] Thus, in a preferred embodiment, a broadening, preferably a statistically significant broadening, of the peak of the distribution function of the hydrodynamic radius of said protein as determined by dynamic light scattering compared to non-denaturing conditions is indicative of denaturing conditions. A preferred measure of the width of said peak is the FWHM.

[0049] In particularly preferred embodiments, a relative increase in said FWHM of at least 50%, at least 40%, at least 30% or at least 20% upon change in physicochemical conditions indicates said change in conditions resulting in denaturing conditions. The term relative increase is defined as follows: (FWHM f -FWHM i )×100 / FWHM i (where the subscripts i and f refer to the beginning (before the conditions were changed) and the end (after the conditions were changed), respectively).

[0050] Any change in FWHM below said value means that in changing conditions, starting from non-denaturing conditions, the new conditions should be subsumed under the term "non-denaturing".

[0051] In a particularly preferred embodiment, the term "non-modifying" applies to a relative increase in FWHM that is below 20%.

[0052] In a further preferred embodiment, said physicochemical conditions are one, more or all of: (i) pH value; (ii) temperature; (iii) concentration of at least one organic solvent; (iv) concentration of at least one salt; (v) concentration of at least one detergent; and (vi) concentration of said protein, polypeptide and / or peptide.

[0053] In a particularly preferred embodiment, the pH change is toward the isoelectric point of the protein, polypeptide and / or peptide to be precipitated onto said heterogeneous surface or solid particulate matter. Proteinaceous molecules are known to be at least soluble near their isoelectric point (IEP). pH-altering agents include, but are not limited to, acids and bases.

[0054] Preferred acids include inorganic acids such as boric acid, sulfuric acid, nitric acid, and hydrogen halides including HF, HCl, and HBr; sulfonic acids such as methanesulfonic acid and benzenesulfonic acid; carboxylic acids such as formic acid, acetic acid, and citric acid; and halogenated carboxylic acids such as trifluoroacetic acid, chloroacetic acid, and fluoroacetic acid.

[0055] Preferred bases include metal oxides, hydroxides, carbonates and bicarbonates. Particularly preferred bases are NaOH, KOH, LiOH, CsOH, Mg(OH)2, Ca(OH)2, Sr(OH)2, Ba(OH)2 and also NH3, tetramethylammonium hydroxide, guanidine, butyllithium and NaH.

[0056] In a particularly preferred embodiment, the change in temperature is a decrease. A decrease in temperature generally reduces the solubility and affects different proteinaceous analytes to different degrees.

[0057] In a particularly preferred embodiment, the change in temperature is an increase. An increase in temperature can result in structural changes, including denaturation, which in turn can promote precipitation.

[0058] In particularly preferred embodiments, the change in concentration of the organic solvent is preferably an increase or decrease of an alcohol, such as ethanol, methanol, n-propanol, i-propanol, etc.; a nitrile, such as acetonitrile; a ketone, such as acetone; a glycol ether, such as 2-methoxyethanol; an aliphatic hydrocarbon, such as hexane; an aromatic hydrocarbon, such as benzene; an aldehyde, such as formaldehyde; or a mixture thereof.

[0059] If the sample to be processed is free or substantially free of organic solvents, increasing the concentration is obviously the preferred option. On the other hand, if the sample to be subjected to precipitation contains organic solvents, for example because it has been subjected to a prior processing involving the addition of organic solvents, reducing the concentration of said organic solvents can also be a means to fine-tune and / or optimize the precipitation conditions.

[0060] In a particularly preferred embodiment, the change in the concentration of the salt is an increase in the concentration of said salt, which is preferably a kosmotropic salt. "Salting out" using kosmotropic salts is known as a means to induce precipitation. Preferred kosmotropic salts include ammonium sulfate, potassium phosphate, and salts with cations selected from Li+, Zn2+ and Al3+ and anions selected from carbonate, sulfate and hydrogen phosphate.

[0061] It is also envisaged to reduce the concentration of the kosmotropic salt. In a further particularly preferred embodiment, the change in salt concentration is a decrease in the concentration of said salt, said salt being preferably a chaotropic salt. Chaotropic salts generally increase solubility. Therefore, a decrease in concentration is preferred. Preferred chaotropic salts are NaCl, MgCl2, guanidine salts such as guanidine hydrochloride, perchlorates such as lithium perchlorate, lithium acetate, barium salts, thiocyanates, iodides and sodium dodecyl sulfate (SDS).

[0062] On the other hand, there may be scenarios in which a high abundance protein has a tendency to precipitate and its presence in large amounts in the precipitate should be avoided, in such cases the addition of chaotropic salts or an increase in their concentration may be desirable.

[0063] The differing effects of different salts / cations / anions on precipitation are known in the art as the Hofmeister series.

[0064] In a particularly preferred embodiment, the change in detergent concentration is an increase in concentration. The addition of detergent is a means to keep in solution proteins, polypeptides and peptides that otherwise have an unnecessarily high tendency to sediment. This applies, for example, to transmembrane proteins such as apolipoproteins.

[0065] To the extent that the sample contains or has been pre-processed by adding a detergent, it is also contemplated to reduce the concentration of detergent, typically as a means to induce precipitation.

[0066] Preferred surfactants are sodium lauryl sulfate (SDS), sodium deoxycholate (SDC), sodium cholate (SC), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton X-100), n-dodecyl-β-D-maltopyranoside (DDM), digitonin, polyoxyethylene(20) sorbitan monolaurate (polysorbate 20), polyoxyethylene(80) sorbitan monooleate (polysorbate 80), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO), polyethylene glycol nonylphenyl ether (NP-40), n-octyl beta-D-thioglucopyranoside (OTG), (2R ,3S,4S,5R,6R)-2-(hydroxymethyl)-6-octoxyoxane-3,4,5-triol (octylglucoside), n-decyl-β-D-maltopyranoside (DM), 3-(4-(1,1-bis(hexyloxy)ethyl)pyridinium-1-yl)propane-1-sulfonate (PPS), sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate (ProteaseMAX), or sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (RapiGest SF); and mixtures thereof.

[0067] Preferred are mild detergents, i.e. detergents that do not denature proteins, polypeptides and peptides, but solubilize them or parts thereof that are hydrophobic and would aggregate in the absence of the detergent. Mild detergents include deoxycholates such as SDS.

[0068] In a particularly preferred embodiment, the change in concentration of said protein, polypeptide and / or peptide is an increase. Such an increase can be achieved, for example, by evaporation.

[0069] In a particularly preferred embodiment, the change in concentration of said proteins, polypeptides and / or peptides is a decrease, for example by adding a solvent such as water or a buffer (e.g. PBS). Preferably, said solvent added mimics the composition of the sample (ignoring said proteins, polypeptides and peptides).

[0070] In a further preferred embodiment, said change in physicochemical conditions is selected from: (i) said increase in temperature, where after said increase the temperature is lower than 50° C.; (ii) said increase in the concentration of an organic solvent, where after said increase the concentration of the solvent is less than 20% (v / v); (iii) said increase in the concentration of a chaotropic salt, where after said increase the concentration of the chaotropic salt is less than 0.5 M; and (iv) said increase in the concentration of a denaturing detergent, where after said increase the concentration of the detergent is less than 0.5% (w / v).

[0071] The above preferred embodiments provide a specific definition of what are more commonly referred to herein as "less harsh" or "non-denaturing" conditions.

[0072] Particularly preferred temperatures after said increase are below 40° C., below 30° C., or not increased at all where it is preferred to maintain the sample at or below ambient temperature (such as 20° C.).

[0073] The term "organic solvent" has its industry-established meaning. It includes both protic and aprotic polar organic solvents. In terms of compound classes, it includes alcohols such as methanol, ethanol and propanol (including i-propanol), aldehydes, ketones such as acetone, and nitriles such as acetonitrile. Additional organic solvents are further disclosed above.

[0074] Particularly preferred concentrations of the organic solvent are less than 15% (v / v), less than 10% (v / v), or less than 5% (v / v), including the absence of organic solvent.

[0075] Preferred chaotropic salts are further disclosed above. Particularly preferred concentrations of said chaotropic salts are less than 0.4M, less than 0.3M, less than 0.2M, less than 0.1M, less than 0.05M or less than 0.01M, including the absence of chaotropic salts.

[0076] Within surfactants or detergents, distinctions can be made with regard to their effect on the structural and functional integrity of proteins, polypeptides and peptides. Denaturing surfactants include sodium dodecyl sulfate (SDS), the avoidance of which or its use at low concentrations is particularly preferred. Another denaturing surfactant is ethyltrimethylammonium bromide. Non-denaturing surfactants include NP40, Triton X-100, Triton X-114, Brij-35, Brij-68, Tween-20, Tween-80, octyl beta-glycoside, octyl thioglycoside, CHAPS, CHAPSO and SDC and their derivatives.

[0077] Particularly preferred concentrations of denaturing detergent are less than 0.4%, less than 0.3%, less than 0.2%, and less than 0.1% (all w / v), including the complete absence of denaturing detergent.

[0078] Due to their less harsh properties, non-denaturing surfactants may be used, preferably at concentrations of less than 5%, less than 4%, less than 3%, less than 2% or less than 1% (all w / v).

[0079] In a more preferred embodiment, reducing agents are not used or, if used, are used at a concentration of less than 0.1% (w / v).Reducing agents include Tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT).

[0080] In a further preferred embodiment, the solid particulate matter is a plurality of microparticles having a diameter of 0.4-500 μm, preferably the plurality of microparticles are identical in composition. More preferred size ranges are 0.5-200 μm, 1-100 μm, or 2-50 μm, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 μm. Particle sizes in a population of particles are generally distributed around an average. The above numbers refer to the average. Particle size can be determined using industry-established methods such as scanning electron microscopy (SEM) or static light scattering (SLS).

[0081] In a preferred embodiment, the amount of particles used is in the range of 0.01-100 μg particles per μl of sample, more preferably 0.1-10 μg / μl or 1-5 μg / μl, for example 2.5 μg / μl.

[0082] In a further preferred embodiment, said rough surface is etched or porous.

[0083] Generally speaking, this applies to both the solid particulate matter as well as the heterogeneous surface, but preferably neither of these are derivatized or functionalized to provide a surface that preferentially binds a particular analyte or class of analytes. This differs from many prior art approaches that use tailored surfaces to enrich or deplete each analyte. In fact, there is no requirement for binding moieties on the surface or the particulate matter. Instead, the effect of the surface and the particulate matter is that of a seed. Seeding is a means to induce a change in the state of a substance where the change in the substance does not occur in the absence of a seed, occurs with a delay, or requires more severe physicochemical conditions to occur.

[0084] In further preferred embodiments, the solid particulate matter consists of or comprises one, more or all of: (i) a magnetic or magnetisable composition; (ii) suspended particles; and (iii) sedimented particles. These various types of particles may be used as a single type of particle when carrying out the methods of the invention, although embodiments are also possible in which more than one type of particulate matter is used, thereby producing multiple first fractions or sediments.

[0085] In a further preferred embodiment, the solid particulate matter consists of or comprises polypropylene (PP), polystyrene (PS), polystyrene divinylbenzene (PS-DVB), poly-tetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyoxymethylene (POM), polyethylene (PE), including high density polyethylene (HDPE) and low density polyethylene (LDPE), polyamide (PA), polycarbonate (PC), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polybutylene terephthalate (PBT), acrylonitrile butadiene styrene (ABS), silica, silanol, ceramic, glass, or metal.

[0086] The preferred materials for said solid particulate matter are also preferred container materials, said container being a roughened container according to the present invention.

[0087] In a further embodiment, said solid particulate matter carries defined moieties, preferably (i) hydrophobic moieties such as C4, C8, C18 and styrene; and / or (ii) hydrophilic moieties, for example hydroxyl, carboxyl, sulfonyl, secondary, tertiary and quaternary amines, preferably hydroxyl, more preferably silanol.

[0088] Specifically, functionalized or derivatized particles of such types are contemplated, although there is no requirement according to the present invention to use them.

[0089] The particles disclosed above are available from a variety of manufacturers and can be prepared by following the guidelines provided in the literature, for example, Hench & West, Chem. Rev. 90, 33-72 (1990); Rahman & Padavettan, J. Nanomaterials, article ID 132424 (2012); Rao et al., J. Colloid and Interface Sci. 289, 125-131 (2005); and Rahman et al., Colloids and Surface A 294, 102-110 (2007).

[0090] The following preferred embodiments of the method of the first aspect relate to preferred downstream processing steps which in turn may precede further downstream analytical methods. A preferred downstream analytical method is MS (typically carried out after one, more than one or all of the processing steps (c)-(j) of the following embodiments).

[0091] Thus, in a preferred embodiment, said processing or fractionation is followed by one or more of the following steps: (c) separating the supernatant from the sediment; (d) washing the sediment and, if necessary, combining a wash solution with said supernatant; and (e) cleaving the proteins, polypeptides and / or peptides in the supernatant of (c) or (d), preferably with a protease, chemically or mechanically. This embodiment focuses on further processing of the supernatant.

[0092] In a further preferred embodiment, said processing or fractionation is followed by one or more of the following steps: (f) separating the precipitate from the supernatant; (g) washing the precipitate; (h) solubilizing or resuspending the proteins, polypeptides and / or peptides from said precipitate; and (j) preferably protease, chemical or mechanical cleavage of the proteins, polypeptides and / or peptides in the eluate obtained in step (h). This embodiment focuses on further processing of the precipitate. Notably, both this embodiment and the preceding preferred embodiment can be used in combination.

[0093] In particularly preferred embodiments, when said solid particulate matter is used, said separating step is carried out by centrifugation, filtration or by magnetic means.

[0094] In a particularly preferred embodiment, when said rough surface is used, said separating step is performed by removing the supernatant, such as by pipetting, draining or decanting.

[0095] In further particularly preferred embodiments, the solubilizing or resuspending step is carried out using a detergent, a solvent such as trifluoroethanol (TFE), a chaotropic agent such as urea or thiourea, an acid such as trifluoroacetic acid (TFA), a base such as NaOH, or a mixture thereof.

[0096] Preferred surfactants are disclosed herein above. In further particularly preferred embodiments, (i) said proteases are selected from serine proteases, serine carboxyproteases, cysteine ​​proteases, aspartic proteases, metalloproteases and metallocarboxyproteases, preferably from trypsin, chymotrypsin, Lys-C, Lys-N, Asp-N, Glu-C and IdeS; (ii) said chemical is HCl, BrCN, BNPS-skatole, formic acid, hydroxylamine or 2-nitro-5-thiocyanobenzoic acid; or (iii) mechanical cleavage is by moving a magnet present in said supernatant or eluate, respectively, at least once.

[0097] With regard to proteases, the classes described are the EC classes as follows: serine proteases (EC 3.4.21), serine carboxyproteases (EC 3.4.16), cysteine ​​proteases (EC 3.4.22), aspartic proteases (EC 3.4.23), metalloproteases (EC 3.4.24), and metallocarboxyproteases (EC 3.4.17).

[0098] Regarding mechanical cutting, preferably by moving a magnet, reference is made to our previous patent application WO2020 / 002577 and EP patent applications Nos. 20174469, 20174484 and 20179317, the entireties of which are incorporated herein by reference.

[0099] Briefly, mechanical cleavage can be performed by a method of cleaving at least one covalent bond in at least one molecule, comprising: (a) colliding at least one magnetic body with said at least one molecule; and / or (b) inducing collision of at least one non-magnetic particle with said at least one molecule by moving said at least one magnetic body.

[0100] Preferred molecules are biomolecules, in particular proteins, polypeptides and peptides. The at least one magnetic body can be implemented as part of a single magnet, i.e. ferrimagnetic, ferromagnetic or paramagnetic material. The magnetic body preferably performs a fluctuating or oscillating motion. Such motion is preferably induced by a fluctuating or oscillating magnetic field. The magnetic field can be generated by a current that fluctuates in turn or oscillates periodically. The amperage of the current as a function of time is preferably a rectangular function. The current preferably flows through a coil that surrounds the vessel in which the mechanical cutting takes place. The vessel contains the sample to be processed and the at least one magnetic body.

[0101] In a further preferred embodiment, the fractions obtained by the method of the invention, i.e. the supernatant and / or the sediment, are subjected to MS, optionally preceded by one, several or all of the intervening processing steps (c) to (j) disclosed above. Suitable MS systems or mass spectrometers include (classified by the analytical device): Time-of-flight (TOF) analyzers, e.g., TIMS-TOF Pro (Bruker) or Synapt XS (Waters), 6230B (Agilent), TripleTOF6600 (Sciex), LCMS-9030 (Shimadzu); Orbitrap Analyzer, e.g., Q Exactive Orbitrap series, Orbitrap Tribrid, Orbitrap Exploris series (Thermo Fisher Scientific); Quadrupole analyzers, such as Altis (Thermo Fisher Scientific), Evoq (Bruker), 6400 series (Agilent), QTRAP (Sciex), SCIEX Triple Quad 7500 (Sciex), Xevo series (Waters), LCMS-8060 series (Shimadzu); and Ion trap analyzer, e.g., LTQ XL (Thermo Fisher Scientific), Amazon series (Bruker), FT-ICR (Fourier transform-ion cyclotron resonance) analyzer, e.g., solariX (Bruker) or LTQ FT Ultra (Thermo Fisher Scientific) Examples include:

[0102] Preferably, a liquid chromatography (LC) device, more preferably a micro- or nanoflow LC system, is preferably coupled on-line with a mass spectrometer.

[0103] In a second aspect, the present invention relates to the use of a vessel with a non-uniform surface and / or solid particulate matter to modulate or enhance the precipitation of a protein, polypeptide and / or peptide. The vessel contains a solution or suspension of said protein, polypeptide and / or peptide. The particulate matter is added to said protein, polypeptide and / or peptide, generally to the solution or suspension.

[0104] Preferred embodiments of the method of the first aspect define preferred embodiments of the use of the second aspect, where applicable.

[0105] In a third aspect, the present invention relates to a solid particulate material having at least one agent attached thereto, said at least one agent being an inducer of precipitation of proteins, polypeptides and / or peptides.

[0106] In preferred embodiments, (i) the at least one agent is a single agent; (ii) the agent is an acid or a base; (iii) the agent is a surfactant, preferably a non-denaturing surfactant; and / or (iv) the agent is a salt, preferably a cosmotropic salt.

[0107] Preferred embodiments of the method of the first aspect define preferred embodiments of the third aspect, where applicable.

[0108] In a fourth aspect, the present invention provides a pre-filled reaction vessel comprising solid particulate matter according to the third aspect.

[0109] Preferred embodiments of the method of the first aspect define preferred embodiments of the fourth aspect, where applicable. [Brief description of the drawings]

[0110] [Figure 1] LoBind is a device used to roughen the inner surface of Eppendorf tubes. [Diagram 2] A LoBind Eppendorf tube with a roughened surface (left) and a regular tube (right). EXAMPLES

[0111] The examples illustrate the invention. Example 1 Seeded Sedimentation Using Particles material and method The sedimentation method on seeding according to the present invention can be used with the iST procedure (Kulak et al., 2014) that utilizes iST buffers and iST cartridges from the iST kit, which can be purchased from PreOmics GmbH (Planegg, Germany), which adds n additional steps before the iST process as described below.

[0112] Step 1 (additional step): In general, as a first step, precipitated seeds, typically 3 μm in diameter if not otherwise mentioned, magnetic beads with silanol surface chemistry, were washed three times with 100 μl of ddH2O and ended with dry seeds. Then, 80 μl of incubation buffer was added, followed by 20 μl of human plasma, roughly corresponding to a protein content of 1000 μg. All components were thoroughly mixed using a vortexer and then incubated at room temperature with mixing at 1,000 rpm for 30 minutes. After incubation, the fluid was separated from the seeds using a suitable separation technique, and then the seeds were washed three times with the incubation buffer described above to obtain dry seeds with proteins precipitated on them.

[0113] Step 2: Then, 50 μl of LYSE-BCT buffer was added, followed by heating and shaking at 95° C. and 1,000 rpm for 10 minutes. After cooling to room temperature, 50 μl of resuspended DIGEST was added and incubated at 37° C. for 60 minutes with shaking at 500 rpm.

[0114] The digestion reaction was quenched by adding 100 μl of STOP buffer. The resulting suspension was mixed thoroughly and transferred onto an iST cartridge, then centrifuged at 3,800 rcf for 1 minute. 200 μl of WASH1 was added, followed by centrifugation as just described. This was repeated as before using WASH2. The flow-through was discarded and the cartridge was transferred to a fresh collection tube.

[0115] 100 μl of ELUTE buffer was added to the cartridge and then centrifuged at 3,800 rcf for 1 minute. This step was repeated once for a total of two elutions, collected in one collection tube.

[0116] The eluate was transferred into an Eppendorf Concentrator under vacuum at 45° C. until completely dry. Typically 10 μl of LC-LOAD buffer from the iST kit was added to resuspend the dried peptides and properly resuspended by shaking and / or vortexing. The concentration of the peptides was measured using a NanoDrop instrument.

[0117] For analysis of protein and peptide identification, the solutions were injected into an LTQ Orbitrap XL mass spectrometer using a Thermo easy nLC1200 liquid chromatography system. For MS measurements, a 45 min gradient from 5% buffer A (0.1% formic acid in water) to 95% buffer B (0.1% formic acid in 80% acetonitrile) was applied in all experiments.

[0118] This general procedure was carried out in several different ways, as shown below: - Experiment 1 (SJ150): The incubation buffer was modified: Sample 1+2: water, Sample 3+4: 0.2x PBS solution, Sample 5+6: 0.4x PBS solution, Sample 7+8: 0.6x PBS solution, Sample 9+10: 0.8x PBS solution, Sample 11+12: 1x PBS solution.

[0119] - Experiment 2 (SJ154): The incubation buffer was modified: Sample 1+2: water, Sample 3+4: 2% ethanol, Sample 5+6: 4% ethanol, Sample 7+8: 6% ethanol, Sample 9+10: 8% ethanol, Sample 11+12: 10% ethanol.

[0120] - Experiment 3 (SJ155): The incubation buffer was modified: Sample 1+2: water, Sample 3+4: 2% acetonitrile, Sample 5+6: 4% acetonitrile, Sample 7+8: 6% acetonitrile, Sample 9+10: 8% acetonitrile, Sample 11+12: 10% acetonitrile.

[0121] - Experiment 4 (SJ156): The incubation buffer was modified: Sample 1+2: pH=2.4, Sample 3+4: pH=4.2, Sample 5+6: pH=6.0, Sample 7+8: pH=6.92, Sample 9+10: pH=9.0, Sample 11+12: pH=10.94.

[0122] - Experiment 5 (SJ164): Incubation buffer pH 10.94 (same as experiment 4, samples 11+12). Here, different seeds were used: Samples 1+2: C18 magnetic beads, Samples 3+4: COOH magnetic beads, Samples 5+6: Tosyl magnetic beads, Samples 1+2: NH2 magnetic beads, Samples 9+10: Protein G magnetic beads, Samples 11+12: Epoxy magnetic beads, Samples 13-15: Internal control (replicates of experiment 4, samples 11+12).

[0123] - Experiment 6 (SJ165): incubation buffer at pH 10.94 (same as experiment 4, samples 11+12). Here, the effect of transferring the seeds to a new flask after the sedimentation step was tested. Samples 1-3: same as experiment 4, samples 11+12 (internal control), samples 4-6: transferring the seeds to a new flask after sedimentation.

[0124] - Experiment 6 (SJ169): Incubation buffer at pH 10.94 (same as in experiment 4, samples 11+12). Here, the influence of the seed porosity was tested. Samples 1-3: Three zeolith beads were used as seeds, the porous material. Samples 4-6: Non-porous ceramic beads were used as seeds.

[0125] - Experiment 7 (SJ174): Incubation buffer pH 10.94 (same as experiment 4, samples 11+12). Here, the effect of the seed surface was tested. Samples 1-3: Glass beads with diameter 100-200 μm were used as seeds. Samples 4-6: Korund F120 with diameter 90-125 μm were used as seeds. Samples 7-9: Silicium F120 with diameter 90-125 μm were used as seeds. These materials are typical spraying processing agents.

[0126] - Control 1 (SJ150, 13-15): iST-BCT procedure as above, but without precipitation (step 1). 2 μl of human plasma was used as educt.

[0127] - Control 2 (SJ130, 1+2; SJ119, 3+4): Step 1 was modified as follows: For samples 1 and 2, 800 μl of Blue Sepharose 6 Fast Flow (Merck, GE17-0948-01) was added to the column containing a polyethylene frit. The liquid from the Blue Sepharose slurry was pushed out using a syringe. Then, 0.8 ml of 0.2×PBS solution was added and pushed out again. This was repeated twice for a total of three times. 64 μl of human plasma was added to the washed Blue Sepharose beads, filled up to 100 μl using 0.2×PBS, and incubated for 1 hour with 360° rotation. Then, 400 μl of 0.2×PBS was added and pushed out using a syringe. The flow-through was collected and dried in an Eppendorf Concentrator. Half of the dried pellet was then processed as described in step 2. For samples 3 and 4, 50 μl of Blue Sepharose beads were washed three times with 100 μl of ddH2O, rapidly vortexed (15 s) and centrifuged (5 s), the supernatant was removed, and equilibrated three times with 100 μl of equilibration buffer (0.05 m KH2PO4 as described by the manufacturer). Then, 50 μl of binding buffer (0.05 m KH2PO4 as described by the manufacturer) was added to the relatively dry beads and carefully transferred onto 10 μl of human plasma. For efficient binding, the beads were incubated for 10 min with shaking at 1,000 rpm. The samples were spun down and the supernatant was transferred to a new reaction flask. 25 μl of 2×LYSE-BCT (PreOmics GmbH) was added and step 2 was performed.

[0128] - Control 3 (SJ134): Samples 1 and 2: 100 μl Protein A magnetic beads (ReSyn Biosciences (Pty) Ltd, South Africa) were washed 3 times with 300 μl PBS using magnetic separation and the fluid was discarded. Then 90 μl of binding buffer (as described by the manufacturer, PBS) was added to 10 μl of human plasma, mixed and added to the washed and dried beads described above. The suspension was incubated for 30 minutes with shaking at 1,000 rpm. After incubation, the supernatant was removed using magnetic separation and added to 100 μl of Protein G beads (ReSyn Biosciences (Pty) Ltd, South Africa), washed and dried as described above. The incubation was performed as above, followed by separation and another round of incubation with Protein G beads. Finally, the supernatant was dried in an Eppendorf Concentrator. If dry, step 2 was performed. Samples 3+ and were treated as above, except that Protein A beads (Magtivio) were used only once.

[0129] result Number of proteins identified

[0130] [Table 1]

[0131] [Table 2]

[0132] Consideration For all experiments shown, a significant increase in protein identification compared to all controls can be observed, demonstrating that precipitation on seeds according to the invention has the advantages disclosed herein above.

[0133] Example 2 Settling using rough surfaces material and method In this example, no particles were used. Instead, a container having a rough surface was used, thereby illustrating an alternative implementation of the invention. Otherwise, the materials and methods are those of Example 1, unless otherwise specified.

[0134] - Experiment 8 (SJ221): The walls of LoBind Eppendorf tubes were roughened using a commercial Dremel instrument (see Figures 1 and 2) and compared with a bead-based workflow (as in Example 1, Experiment 4, Samples 11+12) and with the iST workflow. The last two triplicates serve as controls within this experiment.

[0135] - Experiment 9 (SJ216): Plasma itself was collected using different tubes (Sarstedt Monovette EDTA KE 9ml, Sarstedt Monovette Na Citrate 2.9ml, Sarstedt Monovette Li Heparin 7.5ml, Sarstedt Monovette No Anticoagulant, BD P100 K2EDTA Proprietary Protein Stabilizers 2mL) and centrifuged at 150g for 10 minutes at room temperature. After centrifugation, the supernatant was removed for further processing. Plasma from different donors with the same anticoagulant was pooled. Samples were processed using a bead-based workflow (similar to example 1, experiment 4, samples 11+12) but measured on a Bruker TimsTOF Pro attached to a Thermo easy nLC1200 liquid chromatograph with the same gradient settings as described.

[0136] result The results are shown in the table below. The term "bead-based workflow" refers to Example 1.

[0137] Test 8:

[0138] [Table 3]

[0139] Test 9:

[0140] [Table 4]

[0141] Consideration Experiment 8 shows that rough surfaces increase the number of identified proteins (protein IDs) compared to the regular iST workflow.

[0142] Experiment 9 shows the stabilizing effect of using an anticoagulant to collect plasma.

Claims

1. 1. A method for processing or fractionating a sample containing proteins, polypeptides and / or peptides, comprising: (a) altering the physicochemical conditions of the sample; and (b) (i) and (ii) below: (i) adding solid particulate matter to the sample; and (ii) carrying out the method in a vessel having a roughened surface that is etched or porous. Steps to perform one or both of the following: Including, Steps (a) and (b) may be performed simultaneously or in any order; The method, wherein the processing or fractionation obtains one or more first fractions of proteins, polypeptides and / or peptides as a precipitate on the particulate matter and / or the heterogeneous surface, and a second fraction of proteins, polypeptides and / or peptides remaining in the supernatant, and the physicochemical conditions during the changing step are non-denaturing conditions for the proteins, polypeptides and / or peptides.

2. The method of claim 1 , wherein the sample is a cell lysate or a body fluid.

3. 10. The method of claim 1, wherein step (a), step (b), or both steps (a) and (b) are performed more than once.

4. The non-denaturing physicochemical conditions further comprise: (i) does not include temperatures above 70° C., concentrations of denaturing detergents such as SDS above 0.8% (w / v), and concentrations of organic solvents such as acetonitrile above 25% (v / v); The method of claim 1.

5. The physicochemical conditions are (i) pH value; (ii) temperature; (iii) the concentration of at least one organic solvent; (iv) the concentration of at least one salt; (v) the concentration of at least one surfactant; and (vi) the concentration of said protein, polypeptide and / or peptide. The method of claim 1 , wherein the one, more than one, or all of the following are present:

6. The step of changing the physicochemical conditions comprises: (i) said increase in temperature, the temperature after the increase being less than 50° C.; (ii) said increase in concentration of organic solvent, wherein the concentration of the solvent after the increase is less than 20% (v / v); (iii) the increase in the concentration of the chaotropic salt, wherein the concentration of the chaotropic salt after the increase is less than 0.5 M; and (iv) increasing the concentration of a denaturing surfactant, the concentration of the surfactant after the increase being less than 0.5% (w / v); The method of claim 5 , wherein the compound is selected from the group consisting of

7. 10. The method of claim 1, wherein the solid particulate matter is a plurality of microparticles having a diameter of 0.4 to 500 μm.

8. The solid particulate matter is (i) a magnetic or magnetisable composition; (ii) airborne particles; and (iii) Settling particles The method of claim 1 , comprising one, more or all of:

9. 2. The method of claim 1, wherein the solid particulate matter or the rough surface comprises polypropylene (PP), polystyrene (PS), polystyrene divinylbenzene (PS-DVB), poly-tetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyoxymethylene (POM), polyethylene (PE), including high density polyethylene (HDPE) and low density polyethylene (LDPE), polyamide (PA), polycarbonate (PC), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polybutylene terephthalate (PBT), acrylonitrile butadiene styrene (ABS), silica, silanol, ceramic, glass, or metal.

10. The solid particulate matter is (i) a hydrophobic moiety selected from C4, C8, C18 and styrene; and / or (ii) a hydrophilic moiety selected from hydroxyl, carboxyl, sulfonyl, secondary, tertiary and quaternary amines; The method of claim 1 ,

11. After said processing or fractionation, the following steps are performed: (c) separating the supernatant from the sediment; (d) washing the sediment and combining the wash solution with the supernatant; and (e) protease-, chemically or mechanically cleaving the proteins, polypeptides and / or peptides in the supernatant of step (c) or (d). The method of claim 1 , further comprising one or more of the steps:

12. After said processing or fractionation, the following steps are performed: (f) separating the sediment from the supernatant; (g) washing the sediment; (h) solubilizing or resuspending proteins, polypeptides and / or peptides from the precipitate; and (j) protease-, chemically or mechanically cleaving the proteins, polypeptides and / or peptides in the eluate obtained in step (h). The method of claim 1 , further comprising one or more of the steps:

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