WO2009016431A1

WO2009016431A1 - Sample preparation method and apparatus

Info

Publication number: WO2009016431A1
Application number: PCT/IB2007/004210
Authority: WO
Inventors: Rudiger Hess; Norbert Lamping
Original assignee: Digilab, Inc.
Priority date: 2007-08-01
Filing date: 2007-08-01
Publication date: 2009-02-05

Abstract

A method and apparatus for extracting proteins, peptides and other substances from a sample so as to prepare the sample for subsequent analysis by analytic or diagnostic methods such as mass spectrometry. More particularly, a method and apparatus for preparing a peptide extract from a complex biological sample. The sample containing, among other things, peptides, proteins and salts. The method first filters the sample and subsequently subjects the filtered sample to chromatography. The method is performed using a filtration unit, a chromatography unit, and a unit to hold the final extract of the processed sample, all which fit into one another and finally fit into a centrifuge.

Description

SAMPLE PREPARATION METHOD AND APPARATUS

Field of the Invention

The present invention relates to a method for extracting proteins, peptides and other substances to prepare them for subsequent analysis by analytic or diagnostic methods such as mass spectrometry. More particularly, the invention relates to a method for preparing a peptide extract from a complex biological sample comprising, among other things, peptides, proteins and salts. The process comprises first filtering the sample and subsequently subjecting the filtrate to chromatography. More particularly, the process is performed using a filtration unit, a chromatography unit, and a unit to hold the final extract of the processed sample.

Background Many analytical and diagnostic procedures commence with relatively complex biological samples. The complexity of these samples often interferes with the analytical methods to which these samples are subjected. One reason for this is that the substances to be determined during analysis represent only a minor fraction of the total quantity of the substances present in the sample at the start. Another reason relates to the number of different substances present. As a consequence, the samples often are relatively thick and viscous which, additionally, may result in physical difficulties for the analysis of the sample.

Furthermore constituents which are present in the sample but which are not of interest for the analysis may interfere with the analysis of the substances of interest due, for example, to their large quantities relative to the substances of interest, and due to their properties, such as hydrophobicity, charges characteristics, solubility, molecular size, molecular charges, molecular interaction with affinity or other chromatography resins, molecular interaction with the substances of interest, and/or masking of the substances of interest during the analysis, etc. These factors can interfere with one or more of the analytical steps such as filtration, ultrafiltration, reverse phase, affinity, ion or gel filtration, etc., chromatography, mass spectrometry, enzyme-linked immunosorbent assays (ELISA), radio-immuno assays (RIA), protein chip assays, nucleic acid chip assays, biacore assays, surface-enhanced laser desorption ioπisation (SELDI) assays, etc.

In many cases only certain classes of constituents or individual substances present in the sample are of interest for the analysis. For example, in the discovery of biomarkers such as proteins or peptides or other biomolecules for diagnostic purposes, complex samples such as plasma, serum, whole blood, tissue lysates, cell lysates, or in vitro generated tissue culture supernatants or tissue culture cell lysates are often used. The same is true for the diagnostic determination of the presence or absence and/or quantity of already known biomarkers from such complex biological samples.

Recent studies have indicated that certain proteins, peptides, low molecular weight compounds and the like may be indicators or biomarkers for certain pathological states such as cancer, AIDS, diabetes and cardiovascular and neurological disease or physiological states such as stress, age, pregnancy, subjection to environmental influences such as UV-light, air pollution, noise, ingestion of food, etc. For example Petricoin, et al. (Lancet, 2002, 359:572-577) described the use of peptide patterns and deviations thereof and which was indicated to be a potential method for screening ovarian cancer.

Examples of methods intended to enable the analysis of complex biological samples include WO 98/07036, which describes the analysis of complex biological samples such as plasma or urine without recurring to any hypotheses by sequentially applying for example filtration, chromatography and mass spectrometry. Furthermore US 2003/0199001 describes the combination of commonly known filtration and chromatography methods to prepare a sample for subsequent proteomic applications. Several manufacturers offer individual components, which have some similarity to the components of the device presented in the invention described herein, however these devices represent only parts of the device of the present invention and these parts also differ in their construction from the corresponding parts of the present invention. For example various filtration and small scale chromatography devices are sold by Millipore Corporation, Billerica, MA, USA (Montage Albumin Deplete Kit, ZipPlate™, micro-SPE Plate, MALDIspot Kit, Zip Tip pipette tips with cation resin, Cl 8 resin, C4 resin, metal chelate resin, Centricon Plus, Ultrafree®, Microcon®, Centriplus®, Centriprep®, Centricon®, MultiScreen®, Amicon® Ultra, etc.. ), Varian Inc., Palo Alto, CA, USA, Sartorius AG and VivaScience, Gδttingen, Germany (OMIX Pipette Tips Cl 8 and Cl 8MB, Vivaspin 500), Glygen Corp., Columbia, MD, US (Lab in a Tip, TopTip, Spin Column-in-a-Tip, DiaChromTip), Eppendorf AG, Hamburg, Germany (PerfectPure C- 18 Tips), Qiagen GmbH, Hilden, Germany (various protein purification kits), etc.

Summary of the Invention

In the preferred embodiment of the present invention, a method of preparing a peptide extract from a sample comprising peptides, proteins and salts is described. More specifically, the invention relates to a quantitative method for preparing a peptide extract using a filter device in which a filtrate container of a chromatography device and an eluate container have dimensions such that the components fit within each other. That is, the volume of samples fit into a sample container of the filter device which fits directly into a filtrate container of a chromatography device which finally fits into an eluate container. In a second embodiment, the filtration and chromatography devices are pre- assembled, preferably having a reverse phase chromatography column. The reverse phase chromatography columns are sealed and pre-rinsed so that they are ready for use without the need of prior equilibration and/or washing of the matrix of the chromatography column. In a third embodiment, the method and devices for filtration enable the preparation of a suitable extract from a complex sample and readies the sample for subsequent analysis by, for example, mass spectrometry. This is achieved by sequentially subjecting the complex sample to a filtration step and a chromatography step within a single device comprising at least a filter device, a chromatography device, and an eluate container.

In a fourth embodiment, the device allows a user to prepare samples ready for subsequent analysis, starting from complex samples, which are not very well suited for analysis. Advantages of the device include special features of the construction of the individual components of the device, which allow the preparation of the samples with less handling steps saving work, time and cost, and preventing errors or reducing the numbers potential errors due to elimination of working steps. Furthermore the device requires fewer chemicals for the analysis, as the column is already equilibrated with buffers comprising organic solvents (in the case of a Reverse Phase (RP) column) and is already washed with aqueous buffer. There is no need to prepare, or store these buffers and only the exact amount needed for the column in the device is used during the manufacture of the device. Therefore fewer buffers are wasted and unnecessary amounts of hazardous materials such as organic solvents, chaotropic salt solutions, etc. are avoided.

Another advantage of a device according to the invention is that the user can see whether.the seals, plugs, or caps are removed from the chromatography device, thereby knowing, if the device is new and unused, or whether it has been used previously. This avoids sample contamination. The construction of the device described herein differs in several aspects from known similar devices in the art.

The above and other features of the invention, including various novel details of construction and combinations of parts, will now be more particularly described with reference to the accompanying drawings and pointed out in the claims. It will be understood that the particular device embodying the invention is shown by way of illustration only and not as a limitation of the invention. The principles and features of this invention may be employed in various and numerous embodiments without departing from the scope of the invention.

Brief Description of the Figures

., These and other features, aspects, and advantages of the apparatus and methods of the present invention will become better understood with regard to the following description, appended claims, and accompanying drawings where:

FIG. IA illustrates a non-assembled device according to one embodiment, with a filter device, a chromatography device and an eluate container;

FIG. IB illustrates a sectional close-up view of the chromatography device according to the embodiment illustrated in FIG. IA;

FIG. 2 illustrates a chromatography device sealed at its top with a cap and at its bottom with a twist-off cap; FIGS. 3A to 3E illustrate the steps of a workflow in accordance with a method of the present invention; N FIG. 4A illustrates an assembled device with a sealed chromatography column according to one embodiment of the present invention;

FIG. 4B illustrates the assembled device with a sealed chromatography column as shown in FIG. 4 A and illustrating the application of pressure to break the seals of the chromatography column;

FIG. 5A illustrates the upper and lower spike device of an assembled device in accordance with an embodiment of the present invention;

FIG. 5B illustrates a top view of the upper spike device;

FIG. 6A to 6C illustrate various types of lower spike devices in accordance with an embodiment of the present invention;

FIG. 6D illustrates the device having a lower spike device, as shown in FIG. 6C, positioned within;

FIG. 7 A to 7F illustrate various types of seals for the chromatography column of the chromatography device in accordance with an embodiment of the present invention; FIGS. 8A and 8B illustrate the sealing mechanism of a sample container in accordance with an embodiment of the present invention;

FIG. 9 A and 9B illustrate a cap of the filter device which is modified to form a cartridge holding a buffer and the mechanism and how the buffer comprised in the cartridge is released into the filter device in accordance with an embodiment of the present invention;

FIG. 1OA and FIG. 1OB illustrate a method of the present invention using the assembled device;

FIG. 1 IA to 1 ID illustrate the steps of a workflow in accordance with a method of the present invention; FIG. 12 illustrates a method for the quantitative separation of peptides from salt solutions to enable subsequent quantitative mass spectrometric measurement of peptides;

FIG. 13 illustrates a kit for use in accordance with the method of the present invention; and FIG. 14 illustrates a device for the quantitative preparation of a peptide extract from a sample comprising peptides, proteins and salt in accordance with the present invention.

Detailed Description The present invention relates to a method for extracting proteins, peptides and other substances from complex samples so as to prepare the complex samples for subsequent analysis by analytic or diagnostic methods such as mass spectrometry.

"Complex samples" as used herein refer to samples such as biological samples from living organisms, foods, cosmetics, environmental samples, raw materials, chemicals, etc. Biological samples can originate from all kind of organisms and species such as bacteria, yeasts, viruses, animal, plants, eggs, mammals, rodents, humans, experimental animals such as mice, rats, guinea pigs, rabbits, dogs, cats, pigs, apes, etc. The samples can originate from experimental subjects such as animals or humans, wherein preferably samples from different biological states of the subject are used. For example, pregnant compared to not pregnant, before meals or exercise compared to after a meal or exercise, a healthy subject compared to ill subject, and/or a subject treated with a pharmaceutical or chemical compared to (1) an untreated subject, (2) a subject treated with a different pharmaceutical, (3) a subject treated with a different pharmaceuticals or chemicals, (4) a subject treated with a different dose or (5) a subject treated with a different time schedule, etc.

Examples of biological samples include plasma, serum, whole blood, urine, cerebrospinal fluid (CSF), lymph, sputum, saliva, tear fluid, tissue or cell lysates obtainable, for example, by biopsy, or by surgery (tumor tissue, polyp tissue, fat tissue, muscle tissue, liver tissue, etc.), tissue culture supernatants, cell lysates obtained from in vitro cultured cells. Examples of foods are dairy products, beverages (such as milk), fruit and fruit juice, soda, beer, wine, spirits, etc., pastries, pasta, meant produce, sugars, oils, flour, spices, vegetables, etc. Examples of environmental samples include rain water, ground water, water from rivers, lakes and oceans, sewage, air, emissions, soil samples, plant samples etc. Examples of chemical samples are starting materials, intermediate materials or final products of chemical reactions, medicaments, agricultural chemicals such as pesticides, insecticides and fertilizers, etc., cosmetics such as lotions, soap, make up, beauty creams, hair sprays, deodorants, after shave, perfumes, sun milk, shampoo, etc. , household chemicals, such as detergents, disinfectants, deodorants, etc., industrial or handcraft chemicals such as colors, solvents, fuels, glues, foams, etc. Examples of raw materials are products such as cotton, wool, silk and other natural fibers, natural rubber, leather, unprocessed food such as grains, etc. Chemicals may contain impurities originating for example from starting materials of chemical synthesis, or originating due to storage, due to purification procedures, due to decomposition over time, or representing side products originating for example from the synthesis process, or originating form degradation processes.

With specific regard to the apparatus of the invention, reference is made to FIG. IA, which illustrates an embodiment comprising a non-assembled device 10 that includes an eluate container 12, a chromatography device 14, and a filter device 16. FIG. IB shows the particular components of the chromatography device 14. The chromatography device 14 comprises a filtrate container 18 having a top portion 20 connected to a lower portion 22. The lower portion 22 comprises a chromatography column 24 such as a reverse phase column (RP-Column) having an upper frit 26 and a lower frit 28. The term "frit" as used herein is defined as porous, filamentous, net-like, or sieve-like material or structure for holding the resin or matrix of the column in place.

Column 24 also has a small outlet opening 25 on the end opposite the chromatography device 14. An inlet opening 23 is opposite the outlet opening 25 in column 24. Although not limited in this manner, chromatography column 24, as shown in FIG. IA has an approximate length of greater than 10mm and a diameter of greater than 1.5mm. The sample container 17 of the filter device 16 can have an extension 46 at a lower end, where an upper spike device 30 (shown in FIG. 5A) is connected to the filter device 16. The chromatography column 24 according to the invention can be sealed at the inlet opening 23 and/or outlet opening 25 of the chromatography column 24 to prevent a buffer with which the column 24 is soaked or rinsed, from spilling out of the chromatography column 24 and to prevent the chromatography column 24 from drying out during storage. The structures (shown in Figs. 7A-F) sealing the chromatography column 24 can have various shapes and can seal the column 24 according to different mechanisms. The structures that seal chromatography column 24 (shown in Figs. 7A-F) may be removed, destroyed or broken to open the chromatography column 24 at its inlet opening 23 and/or outlet opening 25. The forces resulting in removal, destruction or breaking of the structures sealing the chromatography column 24 can be, for example, centrifugal forces, pressure subjected to the structures by solid materials, liquids or gas, manual forces subjected to the structures by the operator of the device, etc. The structures sealing the column 24 can work on different principals and can therefore have various shapes such as small "dome-like" structures covering the ends of the column 24. The "dome-like" structures are made from fragile or brittle materials such as thin glass, or the structures can be plugs of various shapes such as conical, cylindrical, etc. which plug up a hole at the inlet opening 23 and/or outlet opening 25 of the column 24, or the structures can be caps which cover openings at the inlet and/or outlet of the column, etc.

As shown in FIG. IB, the column 24 can include chromatography material fixed in a small extension of the filtrate container 18 of the chromatography device 14 between an upper frit 26 and a lower frit 28.The opening at the lower end of the small extension of the filtrate container 18 can be closed and the lower frit 28 being functionally replaced by at least one fine slit (not shown). The slit has a width smaller than the particle size of the chromatography material present in the chromatography device 14. Alternatively or in addition the same kind of slit can functionally replace the upper frit 26 of the column 24.

Exemplary structures suitable to seal the inlet opening 23 and/or outlet opening 25 of the chromatography column 24 are depicted in FIGS. 7 A - 7F (where column 24 has been renumbered as 300, 310, 320 in the various FIGS.). The structures sealing the chromatography column 24 can be made from solid materials, hollow structures, foams, foils, etc., or combinations thereof. The structures sealing the chromatography column 24 can also be made from various materials such as rubber, silicone, glass, metals such as aluminum, iron or copper, plastic materials such as polyethylene, polypropylene, polybutadiene, polystyrol, polyvinylchloride, polytetrafluorethene (Teflon®), polymethacrylic acid ester, polyester, formaldehyde resins, polycarbonate, polyethylene terephthalate (PET), polybutylene terephthalate (PBT), etc. The column chromatography 24 can be sealed at its inlet opening 23 and at its outlet opening 25 by different sealing structures, made from different materials and working on different principals. FIG. 2 illustrates the chromatography device 14 sealed with cap 56 at the top and having a twist-off cap 53 that seals outlet opening 25 at the bottom. The chromatography column 24 can be rinsed with buffer and some buffer 58 is shown in the filtrate container 18. The buffer can be any buffer such as those described in this patent application. The type and composition of the buffer depends on the matrix present in the chromatography column 24 and on the kind of samples to be analyzed. In the case of an RP- column, the buffer can comprise an organic solvent to prepare the column resin, or it can be an aqueous buffer if the column is first prepared with an organic buffer and subsequently with the aqueous buffer. FIG. 3 illustrates the workflow for extracting for example peptides or proteins from a sample using a non-pre-assembled device comprising a filter device 16, a rinse buffer-containing chromatography device 14 and an eluate container 12. As used herein, "peptide" refers to a molecule of covalently attached amino acid residues up to a total molecular weight of 15 kilo Dalton (kDa). The term "proteins" refers to covalently attached amino acid residues with a total molecular weight above 15 kDa. Peptides and proteins may include natural and unnatural amino acids of the L isomeric form or D isomeric form among others, as well as derivatives such as glycosylated, phosphorylated, oxidized, reduced peptides and/or analogues thereof.

For preparing the assembled device 32 (FIG. 3B), cap 56 and twist-off cap 53 at the outlet opening 25 are removed from the chromatography device 14 (FIG. 3A). Filter device 16 is then inserted into filtrate container 18 and the chromatography device 14 is further inserted into eluate container 12. A sample is then added into the sample container 17 of the filter device 16 for centrifugation (FIG. 3B). Following centrifugation the filter device 16 is discarded and eluate container 12 is replaced or emptied (FIG. 3C). An elution buffer is then added to filtrate container 18 (FIG. 3D) and centrifuged again to provide the isolated/extracted peptides for analysis (FIG. 3E).

The type of elution buffer depends on the type of chromatography media used in the column of the chromatography device and on the type of molecules to be eluted from the chromatography media or resins. If for example a reverse phase material is used the elution buffer should contain an organic solvent. If ion exchange material is used the elution buffer should contain appropriate salt solutions with an appropriate pH and appropriate ionic strength for that particular material, similar to the salts described for the washing buffers. Suitable organic solvents used for reverse phase chromatography among others are acetonitrile, methanol, isopropanol, tetrahydrofurane (THF), used as individual solvents or as mixtures such as for example a mixture of 50% volume per volume (v/v) isopropanol, 30 % v/v methanol and 20 % v/v water.

As depicted in FIG. 3, the method involves the use of a chromatography device 14 that comprises a column 24 connected with an inlet opening 23 (shown in FIG. IB) to a filtrate container 18. The column 24 forming part of the chromatography device 14 is pre-equilibrated with buffer and is ready for use. The chromatography device 14 is sealed at the top of filtrate container 18 at its outlet opening 25, for example at the top by a cap 56 (FIG. 2) and at the outlet by a twist-off cap 53. After removal of both caps 53, 56 the column 24 of the chromatography device

14 is placed with its bottom in an eluate container 12 and a filter device 16 is placed into the filtrate container 18 of the chromatography device 14 on top of the chromatography device 14 resulting in an assembled device. Subsequently, sample buffer and/or sample from which an extract is to be prepared, is added into the sample container 17 of the filtration device 16 and the assembled device 32 is subjected to centrifugation.

The filtrate eluting from the outlet opening 25 of the filter device 16 directly enters the filtrate container 18 of the chromatography device 14 and then enters and passes the chromatography column 24 of the chromatography device 14. Finally the flow-through from the column 24 of the chromatography device 14 is collected in the eluate container 12. During this process high molecular weight constituents and insoluble constituents of the sample are retained in the filter of the filter device 16. The substances of interest present in the filtrate of the sample, such as peptides, are bound to the chromatography column 24, and constituents of the sample, or of buffers, such as salts, are in the flow through. The filter device 16 is then discarded, the flow through present in the eluate container 12 is also discarded or the eluate container 12 is replaced by a second eluate container 12a (FIG. 3D). Optionally, a washing buffer is added into the filtrate container 18 of the chromatography device 14 and the assembled device 32 comprising the chromatography device 14 and the eluate container 13 is subjected to a second centrifugation step resulting in washing of the column 24. The flow-through is then discarded from the eluate container 12a or the eluate container is replaced by a fresh one 12b (FIG. 3E). To elute the substances of interest that are bound to the chromatography device 24 (e.g., peptides), elution buffer is added into the filtrate container 18 of the chromatography device 14, and the eluate container 12b is again subjected to centrifugation. The eluate present in the eluate container 12b contains the substances of interest, e.g., the extract of the sample, and is now ready for subsequent analysis by various methods such as, for example, mass spectrometry. The chromatography device 14, prior to its use, can be provided in "dry" form without any buffer in the column 24 (in this case the seals of the chromatography device are only for protection towards for example dust), or can be provided equilibrated with a buffer. The type of the rinse buffer, the sample buffer, the wash buffer and the elution buffer depends on the matrix of the column, the sample and the substances of interest to be extracted from the sample. Examples of suitable buffer-matrix-sample-combinations are given below. Equilibration of the column 24 using buffers comprising an organic solvents, rinse buffers, dilution of the sample with sample buffer and/or washing steps using wash buffer are optional steps. Equilibration of the column 24 in most cases is useful to improve the reproducibility and efficiency of the extraction method. Dilution of the sampie^'using sample buffer is especially useful if viscous samples and/or samples containing high concentrations of proteins, peptides or other substances are used for the extraction. Examples of such viscous samples comprising high concentrations of proteins and peptides are serum, plasma, cell or tissue lysates, tissue culture supernatants containing tissue culture medium with fetal calf serum (FCS), etc. Another reason for using a sample buffer is the introduction of substances into the sample, which help to extract the substances of interest from the sample, for example chaotropic salts such as Guanidine Hydrochloride or Urea help to dissociate proteins and peptides from each other, thereby improving for example extraction of peptides from the sample. Performing washing steps using washing buffer is especially useful, if samples containing high concentrations of proteins, peptides or other substances are used to prepare the extract or if the sample contains substances not binding to the column but which interfere with the subsequent analysis of the extract. v..

FIG. 4A shows an assembled device 32 in accordance with the present invention with a sealed column 24. Here, the three device components of FIG. IA are assembled and the column of the chromatography device is sealed. The filter device 16 comprises a sample container 17 and a filter 34. Chromatography device 14 includes an upper ventilation duct 42 and lower ventilation duct 44. The chromatography device 14 is placed inside the eluate container 12, where the lower frit 28 has a lower seal 38 and the upper frit 26 has an upper seal 36. The eluate container 12 has a lower spike element 39 physically connected to the eluate container 12 and the filter device 16 has an upper spike element 29 physically connected to the filter device. When the filter device 16 is sealed to the chromatography device 14, the pressure breaks both seals 36, 38 of the column 24. The lower spike element 39 penetrates the lower seal 38 and upper spike element 29 penetrates the upper seal 36, as shown in FIG. 4B. Broken seals 36, 38 are indicated in FIG. 4B by the letter "Xi" and "X₂". The term "spike element" is used for spikes forming part of the device, whereas the term "spike device" is used for spikes which are separate parts of the device and have to be assembled into the device. The devices in FIG. 3 do not contain spike elements. Spike elements are necessary, if the column is sealed with structures such as dome-like shaped seals (36 and 38), instead of structures such as plugs or caps — for details on different seals see FIG. 7.

FIG. 5 A shows the detail of an upper spike device 30 and a lower spike device 40. The upper spike element 29 in FIG. 4 is physically connected to the filter device 16. The lower spike element 39 shown in FIG. 4 is physically connected to the eluate container 12. The upper spike device 30 and the lower spike device 40 in FIG 5A-B are not physically connected to the filter device or the eluate container, but are separate, individual parts inserted into the assembled device instead of being already part of the filter device or the eluate container.

The upper spike device 30 comprises an upper spike fixture 200 and a spike element 201. The upper spike device 30 is placed between the filter device 202 and the chromatography device 203 with the spike element 201 pointing into the filtrate container 204 of the chromatography device 203. A top view of the upper spike device 30 is shown in FIG. 5B and illustrates openings 50 present at the bottom of the upper spike fixture 200 which openings allow for filtrate to flow into the filtrate container 204.

The lower spike device 40 has a lower spike fixture 52 provided with ventilation openings 55 and a spike element 205. The lower spike device 40 is placed inside the eluate container 206, and the shape of the lower spike fixture 52 conforms to the shape of the bottom of the eluate container 206 to allow the lower spike device 40 to be placed therein. FIGS. 6A-6D illustrate alternative lower spike devices. FIGS. 6A and 6B illustrate spike devices 100, 101 configured to be received within the lower portion of eluate container 110. The spike device 101 of FIG. 6B includes ventilation openings 115, whereas the spike device 100 of FIG. 6A has a pipe-like structure and therefore at its bottom is open anyway and therefore ventilation openings are not necessary.

The spike device 102 of FIG. 6C as configured has a spike element 108 connected by radius arms 107 to a ring-shaped spike support 109. The spike element 108 in this example is very short compared to the spike elements in FIG. 4 and 5. The ring shaped spike support 109 is positioned within eluate container 112 and may rest upon support rims 61 that extend from the interior sidewalls of eluate container 112 as shown in FIG. 6D. These support rims are only necessary, if a spike device 102 as shown in FIG. 6C is used, and have to be omitted, if spike devices 100, 101, 40 as shown in FIG. 6 A, 6B or FIG. 5, respectively, are used.

Although the spike element 108 in the embodiment in FIG. 6C is shown on the upper surface, the ring-shaped spike support 109 could have a spike on both the upper and lower surface such that a spike element 108 points toward the lower seal 111 of the column 112 regardless in which orientation the ring shaped support is positioned in eluate container 110.

FIGS. 7A-7F illustrate various embodiments of a column showing upper and lower seals, and the subsequent removal or breakage of the seals following. In FIG. 7 A, column 30.0 has upper and lower frits 302 and 303, respectively, being sealed by upper seal 301 and lower seal 304, respectively. FIG. 7B shows seals 301 and 304 being broken by action X| and X₂ after being pierced by spikes (not shown) or broken by other means known to those of skill in the art. This reveals openings 305 and 306 of the upper and lower frits 302 and 303, respectively. FIG. 7C illustrates column 310 having upper and lower frits 312 and 313, respectively, being sealed by upper seal 311 and lower seal 314 in the form of a plug. In FIG. 7D, the plugs 311 and 314 have been removed, such as by centrifugal force revealing openings 315 and 316. FIG. 7E provides column 320 having an upper frit 322 and a lower frit 323 with respective upper seal 321 and lower seal 324 in the form of a cap. The caps are removed in FIG. 7F, such as by centrifugal force to reveal openings 325 and 326. FIGS. 8A and 8B show a cap 401 for the filter device 410. An air tight sealed cap 401 is placed on the top of the filter device 410. In FIG. 8 A cap 401 is provided with an inner sealing mechanism 402. FIG. 8B illustrates cap 401 having an outer sealing mechanism 406. Cap 401 is provided with a septum 400 to allow a syringe or cannula to be inserted there through. When the cap 401 is pushed down a ventilation duct 407 (FIG. 8A) or 408 (FIG. 8B) is opened, allowing the buffer to flow downwards through filter membrane 405 into the chromatography device (not shown), as will be further explained below.

The sample container 403 of the filter device 410 contains a buffer 404 inside, which can be a rinse, wash or sample buffer. Examples of sample buffers include buffers which preserve the sample from degradation, due to for example oxidation, reduction, proteases, phosphatases, lipases and other enzymes, which prevent growth of microorganisms such as bacteria, yeast or fungi, etc. The type of sample buffer depends on the type of chromatography media used in the column of the chromatography device, the type of sample, and the type of molecules to be bound to the chromatography media or resins. For a reverse phase column analysis of peptides or proteins in a sample, sample buffers my include high concentrations of salts such as ammonium salts, guanidine salts, guanidine hydrochloride salts, urea, and other chaotropic salts.

Examples of rinsing or washing buffer include buffered salt solutions which may contain ions such as sodium ions, calcium ions, manganese ions, potassium ions, chloride ions, sulfate ions, phosphate ions, carbonate ions, citrate ions, ammonium ions, Ethylene-Diamine-Tetra-Acetic-Acid (EDTA) ions, Tris-ions, glycine ions, etc.

Buffers such as rinsing buffers, washing buffers, sample buffers, elution buffers may comprise buffer additives that include protease inhibitors, phosphatase inhibitors, kinase inhibitors, lipase inhibitors, glycosylation inhibitors, de-glycosylation inhibitors and inhibitors of other enzymes. The inhibitors preferably are inhibitors which are not proteins or peptides and that do not contain amino acid sequences and preferably have a molecular weight below 1 kDa to prevent them from interfering with the analysis of the sample. Furthermore buffers may comprise agents inhibiting microbial growth such as azide, thimerosal, antibiotics, chaotropic salts, etc. and my comprise further substances such as colorants which for example prevent mixing up of different buffers, pH indicators which signal the pH of the buffer, detergents and other substances, which help to keep the constituents of the sample and/or the buffer itself into solution and which prevent aggregation and/or sticking of substances to surfaces.

Buffers also may comprise "standards" which are suitable to monitor the stability of the buffer itself and/or which are suitable to monitor the stability of the sample and/or the substances of interest present in the samples, such as peptides and proteins. Furthermore the buffers may comprise standards to determine the recovery of the method of the invention. The standards preferably are of the same class of substances as the substances intended to be analyzed in the samples. For example if peptides are to be analyzed in the sample, the standards preferably are peptides, if lipids are to be analyzed the standards preferably are lipids, etc. The standards my be labeled to facilitate their easy determination and/or to distinguish them from the substances being original in the samples. Suitable labels, amongst others, are radioactive and non-radioactive isotope labels, dye labels, fluorophores, luminescent labels, nano particle labels, etc. The washing buffer and the rinse buffer may be the same or different buffers. Sample buffer, washing buffer, rinsing buffer, and elution buffer may comprise anti-coagulants, coagulants, proteases, standards, and other substances.

The invention furthermore envisions the use of standards, which comprise at least one label. The term "label" as used herein, refers to any moiety that functions to: (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the first or second label, e.g. FRET; (iii) affect mobility, e.g., electrophoretic mobility, by charge, hydrophobicity, shape, or other physical parameters, or (iv) provide a capture moiety, e.g., affinity, antibody/antigen, or ionic complexation.

Suitable labels include, for example, radioactive or non-radioactive isotopes such as oxygen-18, nitrogen-15, deuterium, tritium, carbon-14, sulfur-35, phosphorus-32 or phosphorus-33, iodine-125, calcium-45, chromium-51 and the like., isobar labels (e.g. labels which originally have all the same molecular weight, but which upon fragmentation, for example during mass spectrometry, show individually different and distinct mass differences), fluorescent labels such as FITC, Alexa Fluor®, BODIPY, Aminomethylcoumarin (AMCA), Rhodamine, green fluorescent protein, etc., luminescent labels, enzyme labels such as horseradish-peroxidase, alkaline phosphatase, luciferase, galactosidase, etc., toxin labels such as microbial toxins, toxic peptides or toxic small organic molecules, synthetic toxins or toxins used for medical purposes, dye labels such as digoxeginin, metal particles as labels, magnetic particles as labels, polymer particles as labels, biotin or other organic molecules as labels, etc.

FIGS. 9A-C illustrates an alternative embodiment of the present invention with a cartridge 500 (FIG. 9 C), comprising a sealing foil 501 and opposite cap 502 with septum 503. The cartridge 500 is inserted into the sample container 506 and is pre-filled with a liquid 505, such as rinse, wash or sample buffer (FIG. 9 A). When pressure is applied upon cap 502, the cartridge is forced downward such that sealing foil 501 is pierced by filter spikes 507, and at the same time, ventilation duct 508 is opened by releasing the inner sealing mechanism 504 to permit buffer 505 to flow downwards through filter membrane 510 into the chromatography device (not shown).

FIGS. 10 and 11 illustrate an embodiment of the device in accordance with the invention in connection with a method of the present invention. As shown in FIG. 1OA, a liquid 615 is placed inside a cartridge 622 disposed in the filtrate container 614 of the filter device 620, and the sealing mechanism 602 of the cap 601 of the cartridge 622 is opened (Fig. 10B). The column 610 is sealed (607, 611) inside the chromatography device 613, and the column 610 holds a liquid 615, such as a buffer or a buffer comprising organic solvent. A sample container 614 is provided with filter spikes 604 such that when pressure is applied on cap 601 of the cartridge 622 as shown in FIG. 1OB, the sealing foil 603 of the cartridge 622 is pierced and the liquid in the cartridge 622 flows into the chromatography device 613.

FIGS. 3 and 11 depict two exemplarily methods of the invention. While FIG. 3 describes a method working with individual, non-assembled parts of a device, which have to be assembled prior to use, Figure 11 describes a method using a more convenient pre-assembled device, resulting in less handling and working steps for the same method.

As shown in FIG. 1 IA, a sample comprising peptides, proteins and salt is placed by injection through the septum 700 inside the cartridge 713 of the filter device 710, which cartridge is pre-filled with a sample buffer, preferably with a sample buffer comprising a high concentration of a chaotropic salts (FIG. 1 IA). The pre-assembled device 701 is then placed inside a centrifuge. During or prior to centrifugation, pressure placed on the top of the cap 702 causes the upper spike element 705 to pierce the upper seal 706. At the same time, the lower spike element 709 pierces the lower seal 708. This "opens" the column 707 of the chromatography device 711.

During the first centrifugation step the sample buffer and/or sample sequentially passes the filter device 710 and the chromatography device 711. Filtering the sample removes proteins and other high molecular weight materials. The filtrate flows into the filtrate container 704 of the chromatography device 711 and subsequently passes the column 707. The flow-through 723 contains salt, sample buffer and/or buffer present in the column prior to centrifugation and constituents originating from the sample, but which do not bind the column 707. The filter device 710 is removed, and the flow through 723 is discarded from the eluate container 712, or the eluate container 712 is replaced by a new one (FIG. 1 IB). An elution buffer is then added to the filtrate container 704 of the chromatography device 711 and placed together with the eluate container 712 inside a centrifuge for the second centrifugation step. This step elutes constituents of the sample, such as peptides, bound to the column 707 into the eluate container 712 (FIG. 11 C). The resulting peptides in eluate container 712 are then ready for analysis (FIG. HD).

As shown in FIG. 1 IA, the sample was placed by injection through the septum 700 inside the cartridge 713 of the filter device 710 and the cartridge was pre-filled with a sample buffer. But this does not have to be the case. That is, the cartridge 713 of the assembled device 701 does not have to contain sample buffer but the sample may be mixed with sample buffer and then injected through the septum 700 into the cartridge or the sample may be, without any sample buffer, injected into a cartridge 713 not containing sample buffer.

If no pre-filled sample buffer used, the cartridge 713 may be replaced by a cap 702 or there may be neither a cap nor a cartridge combined with the filter device of the pre-assembled device 701. In this case the sample (with or without sample buffer) is directly added into the sample container 703 of the filter device 710. Or if the cap 702 or the cartridge 713 does not contain a septum, the sample buffer and/or sample is added to the sample container 703 or the cartridge 713 by prior removing the cap 702 from the filter device 710.

In FIG. 11 , the spikes may be physically attached to the filter device or the eluate container, or they may represent separate spike devices assembled into the device by placing them either between the filter device and the chromatography device and/or inside the eluate container.

The lower spike elements may be constructed in a way and can be of such dimensions, shape and stability, that they increase the stability of the assembled device, especially during centrifugation, by supporting the chromatography column 707, which may rest with its lower end at the upper end of the lower spike element 709.

Optionally "opening" the column 707 by piercing the upper and/or a lower seal, the upper and/or lower seals are replaced by structures such as caps or plugs, which caps or plugs are removed from the column 707 by for example centrifugal force, gas or liquid pressure. Or, "opening" the column 707 by piercing the upper and/or lower seal by use of a structure such as a spike or the seals can be broken directly by centrifugal forces.

If the seals (such as 706, 708) are made from fragile, brittle material such as thin glass, they can be broken by making use of the weight of the liquids present in the device, pressing on the seals and breaking them during centrifugation. Alternatively, the seals may be broken by manually pushing down by the operator of the device.

In FIG. 11 , the column 707 may not contain any liquid and may not be rinsed with any buffer prior to applying the sample to the pre-assembled device 701. In this case the column might or might not be sealed with an upper and/or lower seal. Furthermore, prior to adding the sample to the sample container 703 of the filter device 710, the assembled device might be washed once or several times by adding wash buffer into said sample container 703 and/or the column 707 might be equilibrated by adding rinse buffer comprising or not comprising an organic solvent into said sample container 703 of said filter device 703 or into the filtrate container 704 of the filter device 711. After centrifugation the flow through 723 in the eluate container 712 is kept in the eluate container 712, or the flow through 723 is discarded, or the eluate container 712 is replaced by an empty new one. And prior to elution of the peptides bound to the column 707, the chromatography device 711 might be washed with washing buffer by placing said washing buffer into the filtrate container 704 of the chromatography device 711 and by performing an additional centrifugation step after which the flow through

723 is discarded or the eluate container is replaced by a new empty eluate container 712. Optionally this washing step might be repeated once or several times. The breaking of the upper and lower seals 706, 708 can occur prior to filling the sample container 703 of the filter device 710 with buffer, sample or mixtures of buffer and sample, or after this step via the next step of centrifuging the pre-assembled device 701. Any suitable structure capable of piercing or breaking the seals 706 and 708 may be used instead of the spikes 705 and 709 described. The invention is not limited to spikes. Any suitable structure for sealing the column 707 may be used instead of the described caps or plugs. The invention is not limited to caps and/or plugs. The column might also be sealed by combinations of seals to be pierced by suitable structures, caps, plugs and other structures suitable to seal the column. For example the top of the column might be sealed by a plug and the bottom by a cap, or the top might be sealed by a plug and the bottom by a seal to be pierced by a spike, etc. hi FIG. 11 , the sample container 703, the cartridge 713 of the filter device 710, the filtrate container 704 of the chromatography device 711 and the eluate container 712 preferably have volume and shape dimensions such that the sample container, cartridge, filtrate container and eluate container fit into the same centrifuge with the same centrifuge rotor. Preferably, the filter device 710, the cartridge 713, the chromatography device 711 and the eluate container 712 are combined in one pre-assembled device 701 prior to the filtering (or dial filtering) step and the chromatography step can be performed in a single centrifuge run without intermediate handling of the sample, the eluate or the devices.

In FIG. 11 , the column 707 can be a column selected from the group comprising reverse-phase (RP) columns, anion exchange columns, a cation exchange columns, affinity columns, hydrophobic interaction chromatography (HIC) columns, hydrophobic liquid interaction chromatography (HILIC) columns, silica, Titanium-dioxide, etc. columns with functional groups comprising a spacer such as a hydro-carbon spacer and a functional group such as alcohol groups (-OH), amine groups (-NR2), sulfite groups (- SO₃H), carbonic acid groups (-COOH), etc. and gel filtration columns. The matrix of column 707 can be replaced by reverse phase or other chromatography resins immobilized into or onto membranes or immobilized at another surface. In FIG. 11 , the filter device 710 can include a vertical or horizontal filter or a filter assembled in an angle between vertical and horizontal. The filter device 710 may include one or more filters. If the filter device has two or more filters, the filters can have the same or different molecular weight cut offs, preferably with the upper filter having a higher molecular cut off.

The filter 710 can have a molecular cut off between 1000 and 1 kDa. For example currently commercially available and suitable filter membranes have molecular weight cut offs of 1, 3, 5, 7, 9, 10, 20, 30, 50, 60, 70, 100, 150, 300, or 1000 kilo Daltons. The filter can comprise a filtration membrane, where the membrane can be fabricated from a material selected from the group comprising regenerated cellulose, cellulose acetate, polyethersulfone, polyvinylidene difluoride (PVDF), and Polytetrafluoroethylene (PTFE), polypropylene or combinations thereof. Furthermore filters can be made from glass and ceramic materials. The filter may be mechanically supported by further membranes made from other materials or may be mechanically supported by other structures. The filter can be used in a fixed angle centrifuge rotor, a swinging bucket centrifuge rotor, or used as a tangential flow filter.

The filtration process can include the application of mechanical pressure applied to the sample by structures such as a piston and/or by pumping liquids or gases into the sample container of the filtration device, or by applying a vacuum to the end of the filtration device pointing to the chromatography device. Preferably the mechanical pressure is provided by centrifugal forces or by the application of gas pressure to the sample container, where the gas includes inert gases, non-inert gases or mixtures of inert and non-inert gases. Examples of inert gases include Argon, Neon, Xenon, Nitrogen, etc. An example of a non-inert gas is Oxygen, and an example of a mixture of inert and non- inert gases is regular air.

In another embodiment, shown in FIG. 12, an apparatus method is provided for the quantitative separation of peptides from salt solutions to enable subsequent quantitative mass spectrometric measurement of peptides. First, a first small container

800 having an extension 801 is provided. The approximate minimal size of the extension

801 can be in the range of 1 Omm height and 1.5mm diameter, with a small opening 802 at the bottom, which at its top is connected to a filtrate container 803 and the small container 800, is filled with reversed phase chromatography material 804. The reverse phase chromatography material 804 can be fixed in the small extension 801 of the first small container 800 using a frit 805. The small opening 802 of the first small container 800 can be closed, and the frit 805 can be replaced by at least one fine slit 806, the fine slit 806 having a width smaller than the particle size of the reverse phase chromatography material 804.

The filtrate container 803 is then filled with a liquid sample 807 having high salt concentrations and comprising peptides, and the first small container is placed into a second container 810. The liquid sample 807 can be ultra filtrated before this step. The filtrate container 803 can have an extension 809 that prevents the small container 800 from falling to the bottom of the second container 810 (an eluate container).

A centrifugal force is then applied to the small container 800 fitted in the second container 808, and the liquid sample 807 passes the reverse phase chromatography material 804 and the peptides present in the liquid sample 807 bind to the reverse phase chromatography material 804. The second container 808 is then exchanged for a new second container 810 or the second container 808 is emptied.

A buffer comprising an organic solvent suitable to elute the peptides bound to the reverse phase chromatography material 804 is then added into the sample container and a centrifugal force is then applied to the first small container 800 fitted into the second container 808, wherein the buffer comprising an organic solvent elutes said peptides from the reverse phase chromatography material 804, and the eluate in the second container 808 is then collected.

Further, a kit, as shown in FIG. 13, is provided for use in accordance with the method of the present invention. The kit 900 can comprise a filter device 901, a chromatography device 902 and an eluate container 903 as illustrated in FIG. 1. The kit 900 can further comprise a combination of a pre-rinsing buffer 904 comprising an organic solvent 906 for pre-rinsing the reverse phase column 905 of the chromatography device 902, an elution buffer 924 comprising an organic solvent for eluting peptides bound to the reverse phase column 905 of the chromatography device, a rinsing buffer 907 to rinse the filter device 901 and/or the chromatography device 902, a washing buffer 908 to wash the reverse phase column 905 prior to eluting bound peptides, test dyes 909, test proteins 910, or other test substances to check the integrity of the filter device 901, standard peptides 911 to check the function of the chromatography device 902 and/or the filter device 901 , and/or to check the recovery and/or reproducibility of the chromatography device 902, and/or to check the recovery and/or reproducibility of the filter device 901, protease inhibitors 912, proteases 913, caps 914 for sealing the sample container 915 of the filter device 901 and/or filtrate container 916 of the chromatography device 902, and/or the eluate container 903, and may also include instructions 917 on how to use the kit.

^• THe protease inhibitors 912 preferably have a molecular weight less than or equal to IkDa. The protease inhibitors function in preventing proteolytic digestion of the peptides and proteins present in the sample, thereby preserving the status of the sample composition as it was at the beginning of the analysis. Preferably the protease inhibitors 912 are not peptides and proteins and lack amino acid sequences to prevent them from interfering with the analysis of the peptides and proteins present in the sample and to prevent that the protease inhibitors mask the peptides and proteins originally present in the sample.

The proteases 913 preferably can be trypsin, and/or chymotrypsin for digesting the peptides and/or the proteins present in the sample. Proteases 913 such as trypsin and/or chymotrypsin are commonly used in the art to digest larger peptides and proteins into smaller fragments, which smaller fragments are more easily detected, quantitatively determined and their sequences are easier to determine using techniques such as mass spectrometry.

The test kit 900 can also comprise tubes 918 for blood collection and/or plasma preparation, where the tubes 918 for blood collection are operated manually or by vacuum. Depending on the intended type of sample, the test kit 900 may contain anticoagulants 919, low molecular weight heparin, citrate, EDTA, Hirudin, Draculin and other anticoagulants. However, low molecular weight, homogeneous in molecular weights (e.g., below 1 kDa) non-protein anticoagulants such as heparin, citrate, or EDTA are preferred for the same reasons that low molecular weight, non-protein protease inhibitors are preferred.

Alternatively if serum is intended as sample type, the test kit 900 may contain clot activators 920 such as factor X activators, and/or glass pearls or other structures to increase the surface thereby facilitating and enhancing the clotting process.

The test kit 900 can also have a water repellent gel, pellet or disc 921 present in the collection tubes, which floats between the blood cell phase 923 and a plasma phase 922. The test kit 900 can also include standard peptides 911 for use in checking the function, recovery and/or reproducibility of the devices. Examples of suitable standard peptides are Substance P, Somatostatin- 14, Neurotensin, Renin Substrate (porcine), ACTH 1-17, Endothelin-1 (amino acid residues 19-38), ACTH (Adreno-Corticotropine) amino acid residues 18-39, beta-Endorphine amino acid residues 6-31, ACTH amino acid residues 1-24, Calcitonin, Insulin B chain oxide (bovine), etc. Alternatively to or in addition to peptides other structures such as micro particles may be used as standards.

Additionally, as shown in FIG. 14, a device for the quantitative preparation of a peptide extract from a sample comprising peptides, proteins and salt is provided in accordance with the present invention. The device comprises a filter device 1001, a chromatography device 1002 and an eluate container 1003. The filter device 1001 includes a sample container 1015 and the chromatography device 1002 includes a filtrate container 1016, and the device includes an eluate container 1003. The sample container 101 S, the filtrate container 1016 and the eluate container 1003 have volume and shape dimensions such that they fit into the same centrifuge with the same centrifuge rotor. The filter device 1001, the chromatography device 1002 and the eluate container 1003 can be combined in one assembled device wherein ultra filtering and the reverse phase chromatography can be performed in a single centrifuge run without intermediate handling of the sample, the eluate or the devices.

The shapes of the filter device 1001, the chromatography device 1002 and the eluate container 1003 are designed in such a way that they accurately fit into each other, that they can be easily assembled together and that they stick together, once assembled together. For this purpose the "connecting surfaces" between the filter device and the chromatography device and also the "connecting surfaces" between the chromatography device and the eluate container are complementary to each other. The purpose of these exactly fitting shapes is to make sure, that the different parts of the assembled device stick to together and especially keep sticking together during centrifugation, preferably regardless whether the assembled device is centrifuged in a fixed angle rotor or in a swinging bucket rotor of a centrifuge.

If for example the lower end of the filter device is conical shaped, the upper part of the chromatography device has a conical opening into which the lower part of the filtering device exactly fits in, if the lower end of the filter device has a hemispherical shape, the upper part of the chromatography device should have a complementary hemispherical opening into which the hemispherical shaped filter device accurately fits in. The lower edge of the filtering device may form a groove, whereas the upper edge of the chromatography device my have a complementary tongue or vice versa, such that the groove and tongue perfectly fit into each other. The grove and tongue can have various shapes such as depicted in Fig. 14. For example the cross section of the grove and tongue could be triangular, quadratic, trapezoid, dentoid, etc. and combinations thereof, which combination might comprise two or more of equal- or different-shaped cross sections.

In principal the same is true for the "connecting surfaces" between the chromatography device and the eluate container and between the "connecting surfaces" between the chromatography device or the eluate container and the spike devices. It is also important, that the individual parts of the device are designed in such a way, that they do not change their relative positions to each other during the centrifugation steps of the methods of the invention. For example the chromatography device has to be of such shape or size, that it cannot drop to the bottom of the eluate container. The lower spike device however should have exactly such a shape and size, that it can drop to the bottom of the eluate container and the spike fixture of the lower spike device should have a "connecting surface" which is complementary to the inner bottom shape of the eluate container. For example if the eluate container has a flat bottom the spike fixture of the lower spike device should also be flat, if the bottom of the eluate container is conical shaped the spike fixture of the lower spike device should also be conical shaped, etc. as shown at the lower right hand side of Fig. 14.

Finally different parts of the assembled device might help each other to stay at their appropriate positions relative to each other. For example the lower spike element or the lower spike device might be of such shapes and sizes, that the lower end of the chromatography, device, e.g. the chromatography column, rests on the upper tip of the spike, which physically supports the chromatography device, especially during centrifugation, and which prevents the chromatography device from dropping into the eluate container, or which for example prevents the chromatography column of the chromatography device from being torn off from the rest of the chromatography device during centrifugation due to centrifugal forces, as these centrifugal forces will have especially strong effects on chromatography column of the chromatography device of the assembled device. FIGS. 1-14 describe many possible embodiments for extracting substances from a biological sample. But the present invention is not limited to these specific embodiments and some of the various embodiments, or parts thereof, may be combined with each. For example, the filter device depicted in FIG. 3 may be combined with the filter spikes and cartridge shown in FIG. 9 and then inserted into the sample container of the filter device shown in FIG. 10. Or, a chromatography device as depicted in FIG. 2 (with a cap on top and a twist-off cap at the outlet of the column) may be substituted with a chromatography device without a cap on top and without a twist-off cap at the bottom, or with a sealed column, as shown in FIG. 7. If a column of the chromatography device is sealed with a dome-like shaped seal at the inlet and/or outlet, the chromatography device can be combined with spike devices as shown in FIG. 5 or 6.

Different combinations of spikes may also be used in each of the embodiments. In FIG. 5B, for example, the spike device may be substituted with a spike element physically connected to the bottom of the filter device, as shown in FIG. 4, element 29. Or, an eluate container with a separate spike device 40, as shown in FIG. 5 A may be used instead with an eluate container with a spike element physically connected to the bottom of the eluate container, as shown in FIG. 4, element 39.

The filter devices of the embodiments may also be interchanged. For example, the filter device comprising a column sealed with a structure such as a dome-like shaped seal at the inlet and/or outlet may be interchanged with a filter device comprising a column sealed with a structure such as a plug and/or cap, without the need of any spike devices to break said seals (FIG. 7C to 7F). Such seals can be removed from the column manually or by centrifugal force.

Prior to the elution of the substances of interest, the column of the chromatography can be washed once or several times with washing buffer by addition of the wash buffer to the filtrate container of the chromatography device and by applying an extra centrifugation step. Again the eluate container may be emptied or replaced by another eluate container.

If the filtration device is not discarded after filtering the sample, but is used to recover proteins, peptides and other high molecular weight substances retained in the filter by the filtration process. The retained substances my be recovered from the filter device by rinsing the sample container with a buffer, and/or the retained substances may be recovered by inverting the filter device and inserting it into a tube and subsequent centrifugation to collect any contents present in the sample container of the filter device into said tube.

If the filter device is rinsed with a buffer containing enzymes or chemicals which cleave the substances retained in the sample container, the cleavage products subsequently can be more easily recovered from the filter device. Preferably this can be done by centrifugation of the filter device and filtering of the cleavage products, which are now small enough to pass the filter of the filter device. Preferably the enzymes in the buffer are proteases and the cleavage products are fragments of proteins or peptides. More preferably the cleavage products are recovered by assembling a new chromatography device and a new eluate container with the filter device containing the cleavage products. In this case an extract from the cleavage reaction can be prepared in the same or in a similar manner, as an extract form the original sample, resulting in a second extract. The second extract preferably contains fragments of proteins and/or peptides/ which originally did not pass the filter of the filter device.

This procedure to recover substances retained in the filter by the filtration process can be used with all methods and embodiments of the instant invention, such as the methods listed in Table 1 below and in the methods of Figures 3 and 11. The second extracts preferably represent fragments of proteins and/or peptides and can subsequently be used for further analysis, preferably by mass spectrometric analysis.

Moreover, as depicted in FIG. 11, a pre-assembled device may be used. The device comprises a filter device with a sample container, which sample container optionally comprises a cap which is a cartridge containing a buffer, preferably a sample buffer, and a septum to inject a buffer and/or the sample into the cartridge, hi addition, the pre-assembled device comprises a chromatography device with a filtrate container and a chromatography column, which chromatography column preferably is a reverse phase column, and which chromatography column preferably is sealed at its inlet and/or its outlet using a suitable structure such as a "dome-shaped" seal, a plug, or a cap as illustrated in FIGS. 7 A - 7F. The chromatography column optionally is pre-rinsed with buffer, in the case of a reverse phase column preferably sequentially, first with a rinse buffer comprising an organic solvent and subsequently with a rinse buffer without an organic solvent, or pre- rinsed in the case of a column different from a reverse phase column only with a suitable, usually an aqueous rinse buffer. In the event that the cap of the filter device is replaced by a cap representing a cartridge preferably with sample buffer in it, the sample is injected into said cartridge, preferably using a syringe, more preferably using a syringe with a needle short enough to avoid piercing the sealing foil at the bottom of the cartridge. The sample is mixed with the optional sample buffer and the pre-assembled device is subjected to the first centrifugation step. Where the filter device does not comprise.a. cap which is a cartridge, the sample container of the filter device is filled with sample buffer and/or sample prior to the first centrifugation step. If the pre-assembled device comprises a chromatography column sealed at its inlet and/or outlet by a "dome-like" seal or a similar structure (see FIG. 7A) and furthermore comprises a structure suitable to pierce or break the seals (see FIG. 4 to 6), the seals are broken prior or during the first centrifugation step. Piercing or breaking the seals is caused by applying a force at the top and/or bottom of said pre-assembled device, for example by manual pressure applied by the operator or by centrifugal forces pushing said structure suitable to pierce or break said seals toward said seals. If the pre- assembled device comprises a chromatography column closed at its inlet and/or outlet with a suitable structure such as for example a plug or cap (see FIGS. 7C to 7F), the suitable structure is then removed from the inlet and/or outlet of said column for example by a centrifugal force and/or by pressure or suction mediated by liquids or gases, preferably inert gases such as Nitrogen, or a noble gas.

If the filter device comprises a cap which cap is a cartridge (FIG. 9C) optionally filled with sample buffer and having a sealing foil at its bottom (see FIG. 9), the sealing foil is pierced by a suitable structure such as filter spikes positioned on top of the filter in the filter device between the filter of the filter device and the sealing foil of the cartridge (see FIG. 9). Piercing of the sealing foil takes place prior to the first centrifugation step by applying a force on top and/or bottom of the assembled device, or the sealing foil is pierced during the first centrifugation step due to the centrifugal forces pushing the sealing foil towards the filter spikes. During the first centrifugation step or during the process of moving the sample by use of liquid or gas pressure through the filter device, and through the chromatography device, high molecular weight and insoluble constituents of the sample are captured in the filter of the filter device, and the filtrate enters the filtrate container of the chromatography device and subsequently the chromatography column. The substances of interest, preferably peptides, bind to the matrix of the chromatography column, preferably a reverse phase column, whereas other constituents of the filtrate such as salts and/or other buffer and sample constituents pass the column and elute as the flow-through into the eluate container.

After the first centrifugation step the filter device is discarded and the eluate container is emptied or replaced by another eluate container. To elute the substances of interest (e.g., peptides) bound to the chromatography column, an elution buffer is put into the filtrate container of the chromatography device and the second centrifugation step is started. If the column used in the chromatography device is a reverse phase column and the substances of interest are peptides, the elution buffer preferably comprises an organic solvent. Optionally, prior to the elution of the substances of interest, the column can be washed once or several times with washing buffer by addition of the wash buffer to the filtrate container of the chromatography device and an extra centrifugation step. Again the eluate container is emptied or replaced by another eluate container.

For clarity, TABLE 1 below lists possible combinations of the different embodiments of devices suitable for use in different variations of the same method in accordance with the instant invention. The devices can be separated into two major groups, pre-assembled devices comprising a filter device, a chromatography device and an eluate container, and a second major group comprising the same three (3) parts but which parts are not pre-assembled (second row in TABLE 1).

Furthermore each of these two major groups comprises either a filter device with an empty sample container, or a filter device with a sample container into which a cartridge is inserted (as shown in FIG. 9 and 10), which cartridge is empty and into which a sample is directly injected, or a cartridge containing sample buffer into which a sample is injected (row 3 in TABLE 1). Each of the so far described 4 groups of types of devices comprises a chromatography device containing a column, which is a reverse phase column (RP) or which is another type of chromatography column (non-RP) such as a ion exchange, affinity, etc. column (row 4 in TABLE 1). Finally these columns forming a part of the chromatography device can be present in the chromatography device without any buffer within the column, or can be present in the chromatography device as an equilibrated column (row 5 in TABLE 1). In the case of reverse phase columns (RP) there is another possible variation. Many types of RP columns require rinsing first with a buffer comprising an organic solvent to enable wetting of the resin of the column. Subsequently the columns have to be equilibrated with an aqueous buffer to prepare them for application of a sample and finally the sample can be applied. Therefore RP columns can be present in the chromatography device in "dry" form without any buffer present inside the column, pre- rinsed with a rinsing buffer, or pre-rinsed with a buffer comprising an organic solvent (requiring an additional rinsing step with buffer during the method to prepare them for application of the sample), or they can be present pre-rinsed with a buffer comprising an organic solvent and subsequently equilibrated with a rinsing buffer (row 5 in TABLE 1). Row 6 in TABLE 1 notes, which figure depict an example of a method using such type of device.

TABLE 1: Methods for use of the device

No. Device Sample container of Chromatography device Method filtration device Colum Buffer described n in figure:

1 Pre-Assembled Empty non-RP no buffer r (FeiInG. 4 A\)

2 rinsing buffer 3 RP no buffer 4 rinsing buffer 5 organic solvent 6 1.organic solvent

2.rinsing buffer

Cartridge containing sample non-RP no buffer

c sαamtvinpiloe, inserted into sample container (FIG. 8 + 9)

8 rinsing buffer FIG.10+11

9 RP no buffer

10 rinsing buffer

11 organic solvent

12 1.organic solvent FIG.10+11 2. rinsing buffer

13 Non-assembled Empty non-RP no buffer (FIG. 1)

14 rinsing buffer FIG. 3+2 15 RP no buffer 16 rinsing buffer 17 organic solvent 18 1. organic solvent FIG. 3+2 2.rinsing buffer

19 Cartridge containing sample non-RP no buffer buffer and/or sample, inserted into sample container (FIG. 9 & 10)

20 rinsing buffer

20 RP no buffer

21 rinsing buffer

22 organic solvent

23 1.organic solvent 2.rinsing buffer

In addition to the different methods listed in TABLE 1, the column of the chromatography device can be sealed or unsealed. If it is sealed, it can be sealed with a structure such as a plug or cap, which is removed from the column during the first centrifugation step. Alternatively, the column can be sealed with a seal, which seal can be broken using a structure such as a spike device or a spike element.

As discussed above, FIGS. 4-6 depict different kinds of spike elements, suitable for use in various types of the device. Spikes, which are physically connected to other parts of the device are just termed "spike elements", whereas spikes forming separate parts of the device, not physically connected to other parts of the devices (see FIGS. 5 and 6) are termed "spike devices". FIG. 7 describes different types of seals for the chromatography column of the chromatography device. Figure 8 describes different designs of the sample cartridge. The different types of spikes, different types of seals for the chromatography column, and different designs for the sample cartridge in principal do not alter the handling steps of methods such as those listed in TABLE 1 but represent different variations how to construct a device suitable for the methods listed in TABLE 1. Not all variations of the design of the different parts of the device are suitable, or do make sense for all methods listed in TABLE 1. Spike structures for example do not make sense for designs comprising columns sealed with caps or plugs. Seals for columns do make less sense for columns not rinsed with buffer, because "dry" columns need not to be prevented from drying out, or from spilling out buffer, but seals can still protect the column from dust and contamination.

Finally a cartridge is used only in some of the methods listed in TABLE 1 and makes more sense, if a buffer is pre-filled in the cartridge. However a cartridge without a pre-filled buffer in it still has the advantage, that a sample can be injected and stored in that cartridge in advance, for example for sample storage purposes, prior to preparing an extract from that sample.

Table 2 shows exemplary dimensions of the chromatography column in the chromatography device, for example, provided that 1 ml plasma is to be processed by the device of the invention and provided that 1 ml plasma is diluted with concentrated Guanidine Hydrochloride solution resulting in a final volume of 4 ml sample with a final Guanidine Hydrochloride concentration of 6 mol/L.

The resulting filtrate passing the filter device typically will contain a total of 2 mg proteins and peptides, depending on the molecular cut-off of the filter present in the filter device. Using a chromatography device having a diameter of the column of 5 mm and a centrifugation time of 2 minutes for passing the sample through the filter device and through the chromatography device, the resulting volume flow rate will be 100 cm/h.

Table 2

Row 1 Row 2 Row 3 Row 4 Row 5 Row 6 Row 7 sample type Total total column required diameter of volume and volume sample protein / material and column a column flow rate volume peptide in protein/pepti volume having a

*.- - after filtrate de binding height of (should be addition of capacity 10 mm 5-400 sample cm/h) buffer

Plasma: 1 4 ml 2 mg Polymer RP 0.2 ml 5 mm 100 cm/h ml 10 mg/ml

Plasma 0.1 0.4 ml 0.2 mg Polymer RP 0.02 ml 0.5 mm 100 cm/h ml 10 mg/ml

Plasma: 0.2 0.8 ml 0.4 mg Silica RP 0.2 ml 5 mm 100 cm/h ml 2 mg/ml

Dependencies of values in Table 2:

Row 2 depends on type of sample, type of buffer, and type of filter (compatibility to buffer) Row 3 depends on type of sample and molecular cut-off of the filter of the filter device Row 4 depends on type of column material; the value of (Row 4 * Row 5) should be equal or bigger than the value in Row 3 to make sure, that protein/peptide quantity does not exceed the capacity of the column; possible column materials are disclosed in the description or as listed in Table 2

Row 5 can be calculated based on Row 3 and Row 4

Row 6 can be calculated based on 5 and the intended column height of 10 mm, for example Row 7 can be calculated based on 6 and the centrifugal or other forces moving the sample through the column and based on the time during which the sample flows through the column The material present in the RP chromatography column preferably contains carbon chains as functional groups, such as C3-C18 (that is, C3, C4, C5, C^ C7, Cg, Cg- C1₀, Cn, C1₂, Ci3, Ci4, Cis, C16, C1₇, and Ciβ), and might also be a material with mixtures of different functional groups. Preferably the chromatography material is made from polystyrene divinyl benzene (PS/DVB). The RP chromatography material preferably has a pore size of about 1000 to 60 angstrom units, preferably about 100-300 angstrom units (preferably, 100-300 angstrom units, more preferably approximately 100 angstrom units, approximately 200 angstrom units, approximately 300 angstrom units), and a particle size of about 5 to 50 micro meter, preferably about 10-30 micro meter; more preferably about 15 micro meter and more preferably about 10 micro meter.. For extraction of peptides below 15 kDa reverse phase (RP) chromatography, hydrophobic interaction chromatQgraphy (HIC) and hydrophobic liquid chromatography (HILIC) are preferably useful.

Experimental

Below are possible experimental conditions for the extraction of peptides from a biological sample according to the various embodiments of the invention:

Experiment 1: Reverse phase chromatography:

If, for example, the sample is human plasma and peptides of up to 15 kDa molecular weight are to be extracted preferably the following conditions and buffers might be used for the chromatography:

- Reverse phase chromatography matrix: Source RPCl 5, General Healthcare, USA Rinse-buffer: water with 0.06 % (w/v) tri fluoric acetic acid (TFA)

Sample buffer: 8 M Guanidine Hydrochloride or 8 M Urea; 25% sample(v/v), diluted with 75 % (v/v) sample buffer, resulting in final 6 M salt concentration

- Washing buffer: water with 0.06 % (w/v) TFA, optional with preferably up to 20 % (v/v) acetonitrile

- Elution buffer: water with 0.05 % (w/v) TFA and 50 to 80, preferably 80 % (v/v) acetonitrile As an alternative to TFA (0.1 - 0.5 % (w/v)), other substances such as formic acid, heptafluor acetic acid, heptafluor butyric acid (0.1 — 0.05 (w/w)), hypochloric acid (about 5mM), NHaSO,*, phosphate buffer, phosphoric acid (0.1 — 0.05M) can be used.

Alternatively to Guanidine Hydrochloride or Urea, other chaotropic salts might be used. The same conditions might also be used for proteins or peptides > 15 kDa.

Alternatively HIC or HILIC chromatography may be used, combined with the use of appropriate buffers.

Experiment 2: Hydrophobic interaction chromatography (HIC):

If, for example, the sample is human plasma and peptides of greater than 15 kDa molecular weight are to be extracted preferably the following conditions and buffers might be used for the chromatography.

HIC chromatography matrix: PLRP-S300, Polymer Laboratories Varian, USA

- Sample buffer: final concentration: 2 M ammonium sulfate (NH4)2SO4, 100 mM sodium di-hydrogene phosphate NaH₂PO.*, pH 7 - Elution buffer: 100 mM NaH₂PO₄, pH 7

- Rinse buffer: 100 mM NaH₂PO₄, pH 7

- Wash buffer: 2 M ammonium sulfate (NH₄^SO₄, 100 mM sodium di-hydrogene phosphate NaH₂PO₄, pH 7

HIC is suitable for peptides up to 15 kDa molecular weight and for peptides and proteins >15 kDa. As buffers for HILIC (hydrophobic liquid chromatography) chromatography the same or similar buffers might be used, which are used for reverse phase chromatography.

Other suitable salts for use in sample, elution, and wash and rinse buffers for HIC are known in the art such as salts comprising as anions: PO₄ ^{(3 >}, SO₄ ^{(2 )}, CH₃COO⁰, Cl^{( )}, Br", NO₃",

C1O₄ ^{{ )}, I", SCN", and comprising as cation NH₄^, Rb⁽⁺⁾, K⁽⁺⁾, Na⁽⁺⁾, Cs⁽⁺⁾, Mg⁽²⁺⁾, Ca^<2+), Ba⁽²⁺⁾. Examples of salts are: Na₂SO₄, K₂SO₄, (NH₄)ZSO₄, Na₂HPO₄, NaCl, LiCl, KSCN. The suitable salt concentration is known in the art and is commonly optimized by empirical testing. Furthermore, depending on the substance of interest to be extracted from the sample, other types of columns might be preferable. For example for extracting carbohydrate or sugar structures affinity matrixes such as lectin columns might be used, for extraction of phosphorylated peptides or proteins affinity columns containing antibodies directed to phospho-thyrosin, phospho-serin and/or phospho-thyrosin might be used. For distinct other classes of ligands, other types of columns might be used such as anion or cation exchange columns.

If the column forming part of the chromatography device is a reverse phase (RP) column the column can be provided "dry", or equilibrated with rinse buffer. Preferably the RP column is first rinsed with rinse buffer comprising organic solvent and subsequently rinsed with an aqueous rinse buffer not containing organic solvent. This is done for example by adding rinse buffer comprising organic solvent into the filtrate container of the chromatography device, assembling the chromatography device with an eluate container, centrifuging the assembled device, adding aqueous rinse buffer not containing organic solvent into the filtrate container of the chromatography device and centrifuging it a second time.

If the RP column is provided with rinse buffer comprising organic solvent, prior to applying the sample to the RP column, the RP column preferably is first rinsed with aqueous rinse buffer without organic solvent, for example by adding aqueous rinse buffer without organic solvent into the filtrate container of the chromatography device, assembling the chromatography device with a eluate container and centrifuging the assembled device. Depending on the type of RP matrix used, a rinse buffer comprising organic solvent as first step to equilibrate the column prior to use of the column might be omitted, or even a "dry" RP matrix without any buffer in it might be directly used, e.g. the sample buffer and/or sample is directly applied to the "dry" RP matrix present in the column.

While there has been shown and described what is considered to be preferred embodiments of the invention, it will, of course, be understood that various modifications and changes in form or detail could readily be made without departing from the spirit of the invention. It is therefore intended that the invention be not limited to the exact forms described and illustrated, but should be constructed to cover all modifications that may fall within the scope of the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method for preparing a peptide extract from a sample, the method comprising the steps of

S (a) filtering said sample to form a filtrate, said filtering being performed using a centrifuge and a filter device with a sample container;

(b) using a chromatography device on the filtrate formed in step (a) to remove salts using said centrifuge and a chromatography device with a filtrate container; and

(c) eluting said peptides from said chromatography device into an eluate container0 using said centrifuge, wherein said filter device in step (a), said chromatography device in step (b) and said eluate container in step (c) have volume and shape dimensions such that said sample container, said filtrate container and said eluate container fit into each other and fit into the centrifuge. 5 2. The method according to claim 1 , wherein the sample includes peptides, proteins and salts.

3. The method according to claim 1 , wherein said filtering in step (a) removes insoluble materials, proteins and/or other high molecular weight materials from the sample. 0

4. The method according to claim 1 , wherein the chromatography device includes a chromatography column.

5. The method according to claim 4, wherein the chromatography column is a reverse phase column.

6. The method according to claim 1, wherein said filtering in step (a) is an5 ultrafiltration or a dial filtration.

7. The method according to claim 6, wherein said ultrafiltration uses a filter having a molecular weight cut off between 1 and 150 kiloDaltons.

8. The method according to claim 1 , wherein said filtrate formed in step (a) has a volume that fits into the filtrate container of said chromatography device of step (b) and the eluate formed in step (b) fits into the eluate container of step (c).

9. The method according to claim 5, wherein said filter device, said chromatography device comprising said chromatography column, and said eluate container are combined in one assembled device wherein the filtering step (a) and the reverse phase chromatography of step (b) can be performed in a single centrifuge run without intermediate handling of any of said sample, said filtrate, said eluate, said devices or said eluate container.

10. The method according to claim 5, wherein said reverse phase chromatography column in said assembled device has an inlet and an outlet, and is supplied soaked with a buffer comprising an organic solvent, and wherein both ends of said reverse phase chromatography column are sealed, said seal being pierced or broken by a spike or other suitable structure positioned in front of each end of said chromatography column when said assembled device is pressed together by applying a force onto the top and/or bottom of said assembled device.

11. The method according to claim 5, wherein said reverse phase chromatography column in said assembled device has an inlet and an outlet, and is supplied soaked with an aqueous washing buffer following prior soaking of said chromatography column with a buffer comprising an organic solvent, and wherein both ends of said reverse phase chromatography column are sealed, said seal being pierced or broken by a spike or other suitable structure positioned in front of each end of said chromatography column when said assembled device is pressed together by applying a force onto the top and/or bottom of said assembled device.

12. The method according to claim 10, wherein said seal is a cap, plug, or other suitable structure, and wherein said cap, plug, or other suitable structure is removable from the front of each end of said chromatography column by centrifugal forces and/or gas or liquid pressure without the need of said seal being pierced by a spike or other suitable structure.

13. The method according to claim 1, wherein said sample container of said chromatography device comprises an extension at said outlet.

14. The method according to claim 10, wherein step (c) in claim 1 is replaced by the following steps:

(d) filling said sample container of said filter device with washing buffer; (e) breaking both seals of said chromatography column and centrifuging said assembled device;

(f) discarding from said eluate container any liquid having passed said chromatography column, or optionally replacing the eluate container;

(g) adding sample buffer and/or a sample into said sample container _ ^•— ^• of said filter device;

(h) centrifuging said assembled device; and

(i) removing said filter device from said assembled device and elute said peptides bound to said chromatography column by adding elution buffer comprising organic solvent to said sample container and by centrifuging said assembled device.

15. The method according to claim 10, wherein step (c) in claim 1 is replaced by the following steps: (d) filling said sample container of said filter device with sample buffer and/or a sample;

(e) breaking both seals of said chromatography column and centrifuging said assembled device;

(f) discarding from said eluate container any liquid having passed said chromatography column , or optionally replacing the eluate container;

(g) removing said filter device from said assembled device and elute said peptides bound to said chromatography column by adding elution buffer comprising an organic solvent into said filtrate container and by centrifuging said assembled device.

16. The method according to claim 10, wherein said liquid having passed said chromatography column is retained in said eluate container rather than discarding it.

17. The method according to claim 10, wherein said spike or suitable structure for piercing or breaking the upper and/or lower seal of said chromatography column is a separate spike device not directly bound to said filter device or eluate container and wherein the upper spike device is positioned between said filter device and/or said chromatography device, and the lower spike device is positioned into said eluate container.

18. The method according to claim 5, wherein said chromatography column is replaced by chromatography material immobilized into membranes or immobilized at another surface.

19. The method according to claim 1 , wherein said chromatography column contains chromatography material fixed by at least one permeable structure such as a frit placed in a column-shaped small extension of said filtrate container.

20. The method according to claim 1 , wherein said chromatography column comprises a opening at the second end (bottom end) of said filtrate container, said opening being closed, and a frit positioned in said opening is replaced by at least one slit, said slit having a width smaller than the particle size of the chromatography material present in said chromatography device.

21. The method according to claim 7, wherein said filter of said filter device comprises a filtration membrane, said membrane fabricated from a material selected from the group comprising regenerated cellulose, cellulose acetate, polyethersulfone, polyvinylidene difluoride (PVDF), and Polytetrafluoroethylene (PTFE) or combinations thereof.

22. The method according to claim 5, wherein the assembled device is provided with a pre-filled washing buffer or sample buffer in said sample container of said filter device, wherein said washing buffer or sample buffer is prevented from spilling out of said sample container of said filter device by a cap positioned over the opening of said sample container.

23. A device according to claim 1, wherein said chromatography devices comprises a reverse phase column, a hydrophobic interaction (HIC) column, a liquid hydrophobic interaction (HILIC) column, an anion exchange column, a cation exchange column, an affinity column, or a gel filtration column.

24. A test kit for use in extracting a peptide from a sample, the test kit comprising: a filter device; a chromatography device; and an eluate container wherein the filter device, the chromatography device and the eluate container have volume and shape dimensions such that the filter device, the chromatography device and the eluate container fit into each other.

25. The test kit according to claim 24, wherein the sample includes peptides, proteins and salts.

26. The test kit according to claim 24, said kit further comprising:

(a) a buffer comprising an organic solvent for pre-rinsing said chromatography column of said chromatography device;

(b) buffer comprising an organic solvent for eluting peptides bound to said chromatography column of said chromatography device;

(c) a rinsing buffer to rinse said filter device and/or said chromatography device; (d) a washing buffer to wash said filter device and/or the chromatography device prior to addition of sample to said filter device and/or prior to addition of filtrate to said chromatography device and/or prior to eluting bound peptides from said chromatography column of said chromatography device; (e) a test substance to check the integrity of said filter device;

(f) standard peptides or other standard substances to check the function of said chromatography device and/or said filter device, and/or to check the recovery and/or reproducibility of said chromatography device, and/or to check the recovery and/or reproducibility of said filter device;

(g) protease inhibitors or other inhibitors preventing degradation of constituents of the sample;

(h) proteases helping to reduce viscosity of the sample; (i) caps for sealing said sample container of said filter device and/or said filtrate container of said chromatography device, and/or said eluate container;

(j) tubes for blood collection and/or plasma preparation; (k) a water repellent gel, pellet or disc, which floats between the blood cell phase and a plasma phase in said tubes according to (j);

(1) an anticoagulant;

(m) instructions to use said kit; and

(n) combinations thereof.

27. The test kit according to claim 26, wherein said protease inhibitors have a molecular weight less than or equal to IkDa.

28. The test kit according to claim 26, wherein said protease inhibitors lack amino acid sequences.

29. The test kit according to claim 26, wherein said proteases are trypsin, and/or chymotrypsin for digesting said peptides and/or said proteins present in said sample.

30. The test kit according to claim 26, wherein said test substance is a test dye or test protein.

31. The test kit according to claim 26, wherein said anticoagulant has a ■ molecular weight less than or equal to IkDa.

32. The test kit according to claim 26, wherein said anticoagulant lacks amino acid sequences.

33. A method for the quantitative separation of peptides from salt solutions to enable subsequent quantitative mass spectrometric measurement of peptides, the method comprising the steps of:

(a) providing a first small container having a top, a bottom and a column-shaped extension, the bottom having a small opening at the bottom, the top connected to a sample reservoir, said small container being filled with reversed phase chromatography material; (b) filling said sample reservoir with a liquid sample having high salt concentrations and comprising peptides;

(c) fitting said first small container into an eluate container;

(d) applying a centrifugal force to said small container fitted in said eluate container, wherein said liquid sample passes said reverse phase chromatography material and said peptides present in said liquid sample bind to said reverse phase chromatography material;

(e) exchanging said eluate container for a new second container or emptying said eluate container; (f) adding into said sample container a buffer comprising an organic solvent suitable to elute said peptides bound to said reverse phase chromatography material;

(g) applying a centrifugal force to said first small container fitted into said eluate container, wherein said buffer includes an organic solvent elutes said peptides from said reverse phase chromatography material; and

(h) collecting said eluate in said emptied or said second container.

34. The method according to claim 33, wherein the column-shaped extension has an approximate minimal size in the range of 10 mm height and 1.5 mm diameter.

35. The method according to claim 33, wherein said sample is subjected to ultrafiltration prior to step (a).

36. The method according to claim 33, wherein said reverse phase chromatography material is fixed in said small extension of said first small container via a frit.

37. The method according to claim 33, wherein said sample reservoir comprises an extension preventing said small container from falling to the bottom of said eluate container.

38. The method according to claim 33, wherein said small opening of said first small container is closed, and said frit is replaced by at least one fine slit, said fine slit having a width smaller than the particle size of said reverse phase chromatography material.

39. A device for preparation of a peptide extract from a sample comprising peptides, proteins and salts, the device comprising: a filter device; a chromatography device; and an eluate container, whereby the filter device, the chromatography device and the eluate container have volume and shape dimensions such that the filter device, the chromatography device and the eluate container fit into each other.

40. The device according to claim 39, wherein said filter device includes a sample container and said chromatography device includes a filtrate container, said sample container, said filtrate container and said eluate container having volume and shape dimensions such that said containers fit into a common centrifuge rotor.

41. The device according to claim 39, wherein said filter device and said chromatography device are combined in one pre-assembled device wherein filtering and chromatography can be performed in a single centrifuge run without intermediate handling of said sample, said eluate or said devices.

42. The device according to claim 39, wherein said chromatography device comprises a sealed, pre-rinsed chromatography column.

43. The device according to claim 39, wherein said filter device comprises a cap and a cartridge, the cartridge sealed with a sealing foil and which cartridge sealed with said sealing foil is pre-filled with a buffer, and which filter device in addition further includes a spike or other suitable structure placed above the filter membrane of said filter device, which spike or other suitable structure pierces the sealing foil of said cartridge if pressed towards said spike or other suitable structure.