Reaction Vessel for Sample Preparation

Abstract

The present invention relates to sample preparation container for purification and/or enrichment of bio-organic compounds from cellular material, viruses and/or sub-components of said cellular material and/or viruses, the container comprising a reaction chamber and a chromatography medium; wherein said reaction chamber is for holding said cellular material, viruses and/or sub-components of said cellular material and/or viruses and is configured such that at least one of the following reactions can be performed therein: lysis, e.g. by sonication and/or boiling; chromatographic purification; reduction; alkylation; and enzymatic reactions such as proteolysis; wherein said chromatography medium is configured to purify and/or enrich said bio-organic compounds; wherein (a) said chromatography medium is located at a wall of said reaction chamber, and said wall is closed or sealed and configured to be opened for obtaining purified and/or enriched bio-organic compounds; or (b) said sample preparation container further comprises a receiving chamber for receiving said bio-organic compounds, said receiving chamber being adjacent to said chromatography medium such that said chromatography medium separates said reaction chamber from said receiving chamber, and the outer face of said receiving chamber is closed and configured to be opened for obtaining purified and/or enriched bio-organic compounds.

Claims

1. Sample preparation container for purification and/or enrichment of bio-organic compounds from cellular material, viruses and/or sub-components of said cellular material and/or viruses, the container comprising a reaction chamber and a chromatography medium; wherein said reaction chamber is for holding said cellular material, viruses and/or sub-components of said cellular material and/or viruses and is configured such that at least one of the following reactions can be performed therein: lysis; chromatographic purification; reduction; alkylation; and an enzymatic reaction; wherein said chromatography medium is configured to purify and/or enrich said bio-organic compounds; wherein (a) said chromatography medium is located at a wall of said reaction chamber, and said wall is closed or sealed and configured to be opened for obtaining purified and/or enriched bio-organic compounds; or (b) said sample preparation container further comprises a receiving chamber for receiving said bio-organic compounds, said receiving chamber being adjacent to said chromatography medium such that said chromatography medium separates said reaction chamber from said receiving chamber, and the outer face of said receiving chamber is closed and configured to be opened for obtaining purified and/or enriched bio-organic compounds.

2. The sample preparation container of claim 1, wherein said bio-organic compounds comprise at least one of the following: proteins; peptides; polypeptides; nucleic acids, e.g. deoxyribonucleic acids and ribonucleic acids; lipids including fatty acids; and metabolites.

3. The sample preparation container of claim 1 further comprising a lid, said lid being adjacent to said reaction chamber and configured to allow feeding of a sample, e.g. by penetration with a needle, and preferably configured to self-reseal after feeding.

4. The sample preparation container of claim 1, wherein the surface of said chromatography medium is configured to act as a filtration surface and/or comprises a further filtration layer and/or reactive layer, said surface or layer facing the interior of said reaction chamber.

5. The sample preparation container of claim 1 comprising a receiving chamber for receiving said bio-organic compounds, said receiving chamber being adjacent to said chromatography medium such that said chromatography medium separates said reaction chamber from said receiving chamber, and the outer face of said receiving chamber is closed and configured to be opened for obtaining purified and/or enriched bio-organic compounds, and further comprising a seal wherein said seal is adjacent to said receiving chamber and configured for obtaining said purified and/or enriched bio-organic compounds.

6. The sample preparation container of claim 1, wherein said container comprises at least one of: polypropylene; polyethylene; a material configured to conduct heat, microwaves, shockwaves, sound waves and/or electricity; a material with low binding surface; a material which is transparent, opaque or selectively transmitting electromagnetic radiation, preferably light.

7. The sample preparation container of claim 1, wherein at least one inner wall of the sample preparation container has at least partially low binding and/or low retention characteristics, preferably by an at least partial coating with polytetrafluoroethylene.

8. A receiving chamber configured to be coupled to the sample preparation container according to claim 1, wherein said chromatography medium is located at a wall of said reaction chamber, and said wall is closed or sealed and configured to be opened for obtaining purified and/or enriched bio-organic compounds and that is configured to receive purified and/or enriched bio-organic compounds.

9. The receiving chamber according to claim 8 comprising a coupling element, which is configured to be coupled to a corresponding coupling element of a sample preparation container wherein said chromatography medium is located at a wall of said reaction chamber, and said wall is closed or sealed and configured to be opened for obtaining purified and/or enriched bio-organic compounds.

10.-11. (canceled)

12. The sample preparation container of claim 1, wherein said chromatography medium comprises (a) at least one of the following materials: reversed-phase materials, cation-exchange materials, anion-exchange materials, mixed-mode ion-exchange materials, ion-complexing materials, an affinity-coupled matrix being antibody-coupled and/or lectin-coupled; and/or (b) two or more stacks of different chromatography media.

13. The sample preparation container of claim 1, wherein (a) said bio-organic compounds comprise one or more proteins, peptides and/or polypeptides and said container is characterized by one, two, three, four, five, six, seven or all of the following (i) to (viii): (i) said container comprises a detergent; (ii) said container comprises a reducing agent; (iii) said container comprises an alkylating agent; (iv) the pH-value in said container is between 7 and 9; (v) said container comprises a standard for mass-spectrometric analysis; (vi) said container comprises a chaotropic agent; (vii) said container comprises an analyte stabilizing chemical; and (viii) said container comprises at least one enzyme selected from proteases; glycosidases; and kinases; and/or (b) said bio-organic compounds comprise nucleic acids and said container is characterized by one, two or all of the following: (i) said container comprises one or more nucleases; (ii) said container comprises reagents for nucleic acid amplification; and (iii) the pH-value in said container is between 7 and 9.

14. The sample preparation container of claim 1, said container further comprising (a) at least one of the following chemicals: milling beads, detergents, chaotropic agents, alkylating agents, reducing agents, organic solvents, standards for mass-spectrometric analysis; and/or (b) at least one of the following enzymes: a protease, a nuclease, a decarboxylase.

15. The sample preparation container of claim 1, wherein (a) said cellular material is one of: intact cells, non-clarified cell lysate, tissue, pathogens; and/or (b) said sub-components are sub-cellular structures, in particular organelles.

16.-19. (canceled)

20. A kit comprising (a) a sample preparation container according to claim 1; and (b)(i) a protease; an alkylating agent; a reducing agent; a standard for mass-spectrometric analysis; a chaotropic agent, a detergent; and/or means for establishing a pH-value in said container of between 7 and 9; and/or (ii) a nuclease; and/or reagents for nucleic acid amplification.

21. The kit of claim 20, further comprising (a) at least one of the following: A bead-milling material, a detergent, a chaotropic agent, an alkylating agent, a reducing agent, an organic solvent, an antioxidant, a UV absorbant, a standard for mass-spectrometric analysis; (b) at least one of the following enzymes: a protease, a nuclease, a decarboxylase, a kinase, a glycosidase; and/or (c) a manual with instructions for performing the method of claim 10.

22. A system comprising (a) a sample preparation container for purification and/or enrichment of bio-organic compounds from cellular material, viruses and/or sub-components of said cellular material and/or viruses, the container comprising a reaction chamber and a chromatography medium; wherein said reaction chamber is for holding said cellular material, viruses and/or sub-compounds of said cellular material and/or viruses and is configured such that at least one of the following reactions can be performed therein: lysis; chromatographic purification; reduction; alkylation; and an enzymatic reaction; wherein said chromatography medium is configured to purity and/or enrich said bio-organic compounds; wherein said chromatography medium is located at a wall of said reaction chamber, and said wall is closed or sealed and configured to be opened for obtaining purified and/or enriched bio-organic compounds; and (b) a receiving chamber according to claim 8.

Description

[0093] The figures show:

[0094] FIG. 1: Exemplary embodiment of a sample preparation container according to the invention. Below is a description of the elements indicated by the reference numerals.

[0095] (100) Sample preparation container.

[0096] (101) A self-sealing rubber lid or rubber plug can be penetrated by a needle or needles to introduce samples, chemicals or shield gas. The rubber plug re-seals after removal of the needle.

[0097] (102) A reaction chamber is constructed for certain reactions such as cell lysis by sonication, protein alkylation, e.g. by iodoacetamide, and proteolysis.

[0098] (103) A surface of the chromatography medium acts as filtration surface. Other surfaces could be applied on top or instead of the filtration surface, for example a non-binding filtration matrix such as microporous Millipore filter.

[0099] (104) A chromatography medium is used to bind, purify and enrich the cellular material of interest. The matrix can be designed to perform multidimensional chromatography which may be effected by using multiple stacks of chromatography media.

[0100] (105) A seal is used to completely seal the reaction chamber before sample lysis. The seal can be opened for washing, purification and elution steps.

[0101] FIG. 2: Exemplary embodiment of a system comprising a sample preparation container and a receiving chamber according to the invention. Below is a description of the elements indicated by the reference numerals.

[0102] (100) Sample preparation container.

[0103] (101) A self-sealing rubber lid or rubber plug can be penetrated by a needle or needles to introduce samples, chemicals or shield gas. The rubber plug re-seals after removal of the needle.

[0104] (102) A reaction chamber is constructed for certain reactions such as cell lysis by sonication, protein alkylation, e.g. by iodoacetamide, and proteolysis.

[0105] (103) A surface of the chromatography medium acts as filtration surface. Other surfaces could be applied on top or instead of the filtration surface, for example a non-binding filtration matrix such as microporous Millipore filter.

[0106] (104) A chromatography medium is used to bind, purify and enrich the cellular material of interest. The matrix can be designed to perform multidimensional chromatography which may be effected by using multiple stacks of chromatography media.

[0107] (107) A wall which is closed and sealed, the wall being configured to be opened for obtaining purified and/or enriched bio-organic compounds.

[0108] (108) Screw thread, e.g. a female screw thread, configured to be coupled with a corresponding screw thread, e.g. a male screw thread.

[0109] (200) Receiving chamber configured to receive said purified and/or enriched bio-organic compounds. The upper side of the receiving chamber may be open or sealed.

[0110] (208) Screw thread, e.g. a male screw thread, configured to be coupled with a corresponding screw thread, e.g. a female screw thread.

[0111] (208a) Sharp-edged extension at the end of the screw thread of the receiving chamber which penetrates the wall of the sample preparation container when the screw thread of the receiving chamber is tightly screwed in the corresponding screw thread of the preparation container.

[0112] FIG. 3: Comparison of the method of the present invention to a published SDC based in-solution protocol. The latter method is the preferred method according to the state of the art; see Leon et al. (Molecular & cellular proteomics 12 (10), 2992 (2013)). Shown are median unique peptide identifications of technical triplicatess.e.m.

[0113] FIG. 4: Quantitative reproducibility of in-depth S. cerevisiae proteome and copy number estimation.

[0114] (a) Frequency of protein identification of four biological replicates. Proteins identified in all four runs are designated as core proteome. (b) MS-signals (Label-free quantification (LFQ) intensities from the MaxQuant output) of five representative proteins spanning the entire dynamic range) in four biological replicates (c) Comparison of LFQ intensities determined in single-shot analysis to LFQ intensities determined in 6-fraction analysis.

[0115] FIG. 5: In-depth coverage of yeast proteomes and estimation of yeast copy numbers, (a), (b) and (c) based on S. cerevisiae dataset. (d) and (e) based on S. pombe dataset.

[0116] (a) Comparison of identified proteins using 6-fraction iST-SCX analysis to the deepest experimental S. cerevisiae proteome (Peng et al., Nature methods 9 (6), 524 (2012)). (b) Correlation of estimated copy numbers using 6-fraction iST-SCX analysis to copy numbers reported using 21 synthetic peptide standards (Picotti et al., Cell 138 (4), 795 (2009)). (c) Distribution of estimated copy numbers. Vertical red line indicates 100 copies per cell. Blue bins represent proteins that are identified uniquely in the 6-fraction iST-SCX analysis, (d) Comparison of identified proteins using 6-fraction iST-SCX analysis to proteins reported in a recent study presenting the deepest proteome of S. pombe (Gunaratne et al., Molecular & cellular proteomics (12(6):1741-51 (2013)). (e) Correlation of estimated copy numbers using 6-fraction iST-SCX analysis to copy numbers reported in another recent in-depth analysis of S. pombe (Marguerat et al., Cell 151 (3), 671 (2012)).

[0117] The following examples illustrate the invention but should not be construed as being limiting.

EXAMPLE 1

Purification of Peptides for Proteomics Analysis Using Mass Spectrometry

[0118] The system has been tested in the field of proteomics using completely sealed tubes or tubes only sealed at one side (usually the bottom, the bottom being the location of the receiving chamber or where the receiving chamber may be attached, respectively). Cells were lysed within the described tube as well as outside the tube without any notable disadvantages for in-tube lysis. Contained proteins were proteolytically digested and crude contaminants were filtered on the surface of the reversed-phase purification matrix C18, which was embedded in Teflon material. The peptides were enriched and desalted on the C18 material. Clean peptides were eluted from the C18 material and subsequently analyzed by LC-MS/MS.

[0119] The results demonstrate high stability and the ability to scale up or down in terms of sample quantities. The efficiency was highly increased, such that no loss was observed. The efficiency of the approach enables the processing of few single cells, which was not possible by established state of the art techniques as shown by processing of as few as 500 HeLa S3 cells. The processing speed is highly increased compared to current methods with a complete sample preparation within 1 h time compared to at least approximately 4 h time, or more commonly 12 h, with other state of the art methods. Expert knowledge is not required because every step is simplified and the source for failure is reduced to a minimum. Very few unwanted modifications of analytes (oxidation, modification by UV light etc.) were observed. Reproducibly high identification rates were observed by LC-MS analysis (MS/MS ID rate of 60%). The amount of contaminations was significantly reduced, also when working with low quantity samples. Polymers or plastics may occur as source of contamination, owing to one or more transfer steps as required for previously known procedures. Sample preparation may involve working with harmful and poisonous chemicals such as DTT and iodoacetamide, which can be provided in a pre-filled and sealed container of the invention, thereby reducing the risk of harm. The separate materials, which could be used to produce the reaction vessels are well known and described and could be combined in a very cheap manner. An automated version of the system is feasible and can easily be implemented.

EXAMPLE 2

Copy Numbers in S. Cerevisiae and S. Pombe

[0120] Protein copy numbers are of great interest to the biological and systems biological communities and we reasoned that a streamlined, minimalistic sample processing method could provide particularly unbiased values. To evaluate the minimalistic sample processing method on the well characterized yeast model system we grew S. cerevisiae in four biological replicates and processed them in parallel in the 96-well format (100 uL culture at OD.sub.600=0.8). Four single-run analyses together identified 4,270 distinguishable protein groups and remarkably, 97% of them were detected in at least three of the four replicates with high quantitative reproducibility (Total median sequence coverage: 34.4%, total number of unique peptide IDs: 46,125; FIG. 4a, b). In our recent yeast proteome analysis in single-run mode, which used the same downstream LC-MS/MS set-up (Nagaraj et al., Molecular & cellular proteomics: MCP 11 (3), M111 013722 (2012)), mean identification in each individual run were 4,084 protein groups (33,122405 sequence-unique peptide identifications, median seq. coverage 23.4%), whereas the minimalistic processing method produced an even higher numbers (4,144 protein groups, 37,8801771 sequence-unique peptides, median sequence coverage 27.2%).

[0121] SCX-fractionation of a yeast sample directly from the reaction device into six autosampler vials, followed by essentially the same LC-MS/MS analysis as before, quantified 4,577 protein groups, the largest expressed yeast proteome reported to date. Importantly, we did not identify any of the 656 dubious open reading frames, which are thought not to represent expressed messages or proteins. Excellent correlation of label-free intensity values with those of a single-run analysis (R.sup.2=0.91), shows that in-StageTip fractionation did not introduce any biases, even in the very low intensity region (FIG. 4c).

[0122] The deepest previous proteome of exponentially growing S. cerevisiae used five different proteolytic enzymes as well as extensive, column-based SCX fractionation of peptides (Peng et al., Nature methods 9 (6), 524 (2012)). Our single six-fraction dataset largely encompassed the previous study (94.9%) and added 400 proteins, among which intrinsic membrane proteins were significantly enriched (p=9.4106) (FIG. 5a). We next used the label-free MS-signal for each protein as a fraction of the total MS-signal of the proteome (Wisniewski et al., Molecular systems biology 8, 611 (2012)) to estimate copy numbers for 4,570 yeast proteins. Copy numbers have previously been established for 21 yeast proteins using synthetic peptide standards 6 and our values agree well within the expected uncertainties (R.sup.2=0.82) (FIG. 5b). The most abundant yeast protein, at 1.610.sup.6 copies per cell, was the glycolytic enzyme Tdh3p, which is encoded in three genomic loci. The median yeast protein had approximately 800 copies per cell and a copy number range of a factor of 2000 contained more than 90% of the proteins. The six ORC complex members have a median copy number of 332150, an interesting relation to the estimated 500 origins of replication in S. cerevisiae (Nieduszynski et al., Nucleic acids research 35 (Database issue), D40 (2007)).

[0123] We were intrigued that more than 763 yeast proteins had less than 100 copies per cell (FIG. 5c), a much larger proportion than in a classical study of yeast copy numbers (Ghaemmaghami et al., Nature 425 (6959), 737 (2003)). This population was significantly enriched for the GO terms cell cycle process and DNA repair (p<910.sup.10 and <1.810.sup.4, respectively).

[0124] For very low abundance proteins, a weak MS signal may introduce uncertainties, nevertheless we measured largely consistent copy numbers for members of the anaphase promoting complex (APC), indicating about 30 APCs per cell. Proteins only present in certain cellular states were often found with very low apparent copy numbers such as the cyclin CLB2 (G2/M phase), at 100 copies or the kinase inhibitor FAR1 (G1 phase) at about 50 copies. This illustrates that our dataset already includes contributions from several different proteomic states.

[0125] S. pombe diverged from S. cerevisiae more than 400 million years ago and provides an interesting comparative model. The deepest proteomic study of that organism very recently employed several growth conditions and very extensive, orthogonal fractionation to identify 3,542 proteins (Gunaratne et al., Molecular & cellular proteomics: MCP (2013)). Using the six-fraction approach on exponentially growing cells only, we obtained 4,087 proteins searching against the same database. This represents 80% of S. pombe ORFs and covers 96.5% of the previous proteome as well as 670 additional, generally low-abundance ones (FIG. 5d). In reference to another deep S. pombe proteome (Marguerat et al., Cell 151 (3), 671 (2012)), our S. pombe copy numbers agreed very well with those reported for 34 proteins for which isotope labeled standards had been synthesized (R.sup.2=0.89) and there was no apparent bias against any protein class, including intrinsic membrane proteins (FIG. 5e). The most abundant proteins had around 10.sup.6 copies per cell, similar to S. cerevisiae, but the proportion below 100 copies was much reduced (17% vs. 3%). Median copy number was 5,137, about six-fold higher than in S. cerevisiae. The lowest expressed 5% of the proteome was significantly enriched for replication fork processing and DNA repair related proteins (p<1.110.sup.6 and <1.210.sup.6, respectively). This fraction of the proteome contains many so far uncharacterized S. pombe ORFs (59 of 207 proteins; p<3.910.sup.5).