SOLID MIXTURE COMPRISING STANDARD PROTEIN

Abstract

Disclosed are a mixture comprising at least one internal standard protein, a container comprising the mixture, a method for preparing a container with the mixture, a method for determining the amount of a target protein present in a sample, providing a container comprising the mixture, as well as a kit for carrying out the methods.

Claims

1. Solid mixture comprising: at least one internal standard protein; at least one chaotropic agent or a derivative or salt thereof; and optionally a buffer.

2. Solid mixture according to claim 1, wherein said chaotropic agent is selected from the group consisting of urea, guanidine, thiourea and derivatives and salts thereof.

3. Solid mixture according to claim 1, further comprising a sample suspected to comprise at least one target protein.

4. Solid mixture according to claim 3, wherein said internal standard protein comprises a fragment of said target protein.

5. Method for preparing a container comprising a solid mixture comprising at least one internal standard protein, the method comprising: providing a solution comprising said at least one internal standard protein and at least one chaotropic agent selected from the group consisting of urea, guanidine and derivatives and salts thereof, placing said solution in a container, removing residual liquid from said solution, thereby obtaining a container comprising a solid mixture comprising said at least one internal standard protein and said at least one chaotropic agent.

6. Method according to claim 5, wherein said chaotropic agent is selected from the group consisting of urea, guanidine, thiourea and derivatives and salts thereof.

7. Method according to claim 5, wherein said chaotropic agent is present in said solution in a concentration of at least 0.5 M, such as at least 1 M, such as at least 2 M, such as at least 3 M, such as at least 4 M, such as at least 5 M, such as at least 6 M, such as at least 7 M, such as at least 8 M.

8. Method according to claim 5, wherein the step of removing residual liquid from said solution comprises removing liquid by means of reduced pressure.

9. Container comprising a solid mixture according to claim 1.

10. Method for determining the amount of a target protein present in a sample, the method comprising: providing a container according to claim 9; unless already present, adding a sample suspected of comprising at least one target protein to said solid mixture, thereby preparing a test sample, subjecting said test sample to analysis, using the results of the analysis to determine the amount of said at least one target protein in said sample by comparison with said internal standard protein.

11. Method according to claim 10, wherein said internal standard protein comprises a fragment of said target protein.

12. Method according to claim 10, wherein the method further comprises a step of long-term storage of said sample preceding the steps of subjecting said sample to analysis and determining the amount of said at least one target protein in said sample by comparison with said standard protein.

13. Method according to claim 10, wherein said sample is a bodily fluid sample selected from the group consisting of plasma, serum, blood, cerebrospinal fluid, dry blood spots, saliva and urine.

14. Kit for carrying out the method according to claim 10, the kit comprising: a container according to claim 9, and instructions for carrying out the method.

15. Container comprising a solid mixture obtainable by a method according to claim 5.

16. Method for determining the amount of a target protein present in a sample, the method comprising: providing a container according to claim 15; unless already present, adding a sample suspected of comprising at least one target protein to said solid mixture, thereby preparing a test sample, subjecting said test sample to analysis, using the results of the analysis to determine the amount of said at least one target protein in said sample by comparison with said internal standard protein.

17. Method according to claim 16, wherein said internal standard protein comprises a fragment of said target protein.

18. Method according to claim 16, wherein the method further comprises a step of long-term storage of said sample preceding the steps of subjecting said sample to analysis and determining the amount of said at least one target protein in said sample by comparison with said standard protein.

19. Method according to claim 16, wherein said sample is a bodily fluid sample selected from the group consisting of plasma, serum, blood, cerebrospinal fluid, dry blood spots, saliva and urine.

20. Kit for carrying out the method according to claim 16, the kit comprising: a container according to claim 15, and instructions for carrying out the method.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG. 1 schematically shows that the tested isotopically labeled internal protein standards represent a stretch of amino acids which is unique for the target protein of interest, and are fused to a tag sequence, denoted Tag-heavy, which is used for quantification of the internal standard protein by comparison with an identical sequence, denoted Tag-light, which is not isotopically labelled.

[0066] FIG. 2 illustrates the workflow used to estimate the effect of vacuum drying internal standard proteins as compared to internal standard proteins kept in solution, as well as the effect of room temperature storage on the stability of vacuum-dried internal standard proteins.

[0067] FIG. 3 shows extracted chromatograms, showing overlaps of the areas under the curve of a peptide resulting from trypsin digestion of the Tag-heavy and Tag-light polypeptide sequences.

[0068] FIG. 4 shows the result of a comparison of median values from triplicate digestion and Tag-based quantification of isotopically labeled standard proteins that were kept in solution and of isotopically labeled standard proteins that were vacuum dried according to the present disclosure.

[0069] FIG. 5 shows a density plot of CVs between the quantification results of all vacuum dried isotopically labeled protein standards stored at room temperature for 0 (median of the triplicate), 1 and 4 weeks, as illustrated in FIG. 2.

[0070] FIG. 6 illustrates the workflow used to estimate the quantification precision of 100 proteins subjected to different digestion times, using a mixture of 100 vacuum dried internal standard proteins.

[0071] FIG. 7 shows the results of cluster analysis, exhibiting the same digestion efficiency for both endogenous proteins and internal standard proteins according to the disclosure for most peptides, i.e. the peptides of Cluster 2 and Cluster 4.

[0072] FIG. 8 shows the technical reproducibility of quantification as coefficient of variation between three technical replicates per peptide of every time point with medians (indicated in the figure) ranging from 4.6% to 6.1%.

[0073] FIG. 9 shows all quantified proteins using the mixture of vacuum dried internal standard proteins and their dynamic range, as measured after 16 hours of digestion.

EXAMPLES

Example 1

Stability Assessment of Vacuum-Dried, Isotopically Labeled Protein Standards

Materials and Methods

[0074] 96 internal standard proteins were randomly selected from an in-house produced library of stable isotope-labeled internal standard proteins, and aliquots thereof were individually added to a 96-well plate. Subsequently, quantification tag (Tag-light, absolutely quantified by amino acid analysis) was diluted to a final concentration of 10 M in 1x PBS (phosphate buffered saline) and 1 M urea, and aliquoted to another 96-well plate. FIG. 1 illustrates the relationship between the endogenous protein, the internal standard protein, the Tag-heavy sequence and the Tag-light sequence. Aliquots from the plate with internal standard proteins were distributed into 8 new plates, so that every well contained 5 l (50 pmol) of each internal standard protein. Five of the eight plates were vacuum dried at 42 C. for 3 h and stored at room temperature. During that time, the remaining three plates were kept on ice with the internal standard proteins in solution.

[0075] Digestion: Three of the five vacuum dried plates were prepared together with the three plates of aliquoted standards kept in solution. Prior to the addition of 5 l of 10 M Tag-light, 55 l of 90.9 mM ammonium bicarbonate (ABC) were added to each well of the 96-well plates with vacuum dried internal standard proteins and 50 l of 100 mM ABC were added to each well of the 96-well plates with internal standard proteins that were kept in solution. All six plates were sonicated for 180 s and digested by addition of 200 ng of porcine trypsin (Thermo Scientific) overnight at 37 C. Digestion was quenched by addition of formic acid (FA) to a final concentration of 0.6% (v/v) and samples were analyzed using LC-MS/MS operating in parallel reaction monitoring (PRM) mode.

[0076] The remaining two of the five vacuum dried plates were kept at room temperature for 1 and 4 weeks, respectively, prior to addition of ABC and Tag-light, trypsin digestion and LC-MS/MS-PRM analysis as described above. The workflow is illustrated in FIG. 2.

[0077] Quantification of Internal Standard Proteins Usinq LC-PRM:

Quantification was performed using an Ultimate 3000 LC online system (Thermo Fisher) connected to Q Exactive HF MS (Thermo Fisher). 2.5 pmol of each internal standard protein was loaded onto an Acclaim PepMap 100 trap column (cat. no. 164535; Thermo Scientific), washed 3 min at 8.5 l/min with Solvent A (3% acetonitrile (ACN), 0.1% FA and then separated by an analytical PepMap RSLC C18 column (cat. no. ES802; Thermo Scientific). A linear 3-min gradient ranging from 3% to 20% Solvent B (95% ACN, 0.1% FA) at 0.6 l/min was used for eluting the peptides. The analytical column was then washed for 3 min at 1 l/min with 99% solvent B and re-equilibrated with 3% B at 0.6 l/min for 4 min.

[0078] The MS operated in PRM mode with each cycle comprising one full MS scan performed at 15,000 resolution (AGC target 2e5, mass range 350-1,600 m/z and injection time 55 ms) followed by 20 PRM MS/MS scans at 15,000 resolution (AGC target 1e6, NCE 27, isolation window 1.5 m/z and injection time 105 ms) defined by a scheduled (0.4 min windows) isolation list.

[0079] Resulting RAW files were loaded into Skyline (v. 20.1.0.76; MacLean et al. (2010), Bioinformatics 26:966-968) together with sequences and transitions of Tag-light and Tag-heavy. Chromatograms for transitions of peptide DLQAQWESAK (SEQ ID NO:1; Table 1) were extracted from the RAW files and areas under the respective curves calculated as shown in FIG. 3 for five examples. The ratios between heavy and light Tag sequence peptides were exported, and the ratios obtained with standard proteins in solution were plotted against those obtained using vacuum dried internal standard proteins according to the present disclosure (FIG. 4).

Results

[0080] The ratios of areas under the curve for light and heavy Tag peptides obtained using the isotopically labeled internal standard proteins that were kept in solution were plotted against the corresponding ratios obtained using isotopically labeled internal standard proteins that had been vacuum dried (FIG. 4). The results demonstrate that there is no difference between the two strategies and that the amount of accessible, isotopically labeled standard protein is the same, whether the isotopically labelled standard was kept in solution or vacuum dried and solid prior to addition of sample to be analyzed and enzymatic digestion.

[0081] Furthermore, plates stored at room temperature over extended periods of time in the vacuum dried format (1 and 4 weeks) showed a retained stability and accessibility of the isotopically labeled protein standards, as evidenced by the coefficients of variation (CVs) below 20%. This is shown in FIG. 5. Only one isotopically labeled standard protein exhibited a CV above 20% (CV=29.1%). For this variant, the internal standard protein and its Tag-heavy sequence were 100 times less abundant than the added Tag-light quantification tag, which caused the decreased precision of quantification and suggests that production of this isotopically labeled standard protein wasn't entirely successful. In fact, the CV of triplicate quantification of the same isotopically labeled standard protein kept in solution was 36.8% (data not shown), which supports the hypothesis that the decreased precision of quantification between the three time points was caused by the big difference in amount (large off-ratio) between the Tag-light and the Tag-heavy peptides and that the accessible amount of this internal standard protein remained the same.

[0082] Decay of the Tag-light quantification tag due to repeated freeze-thaw cycles was observed, but was constant over all replicates and could therefore be normalized for. The normalization for constant Tag-light degradation over the three repeated freeze-thaw cycle is reasonable, since it is not possible for the amount of internal standard protein to increase over time.

TABLE-US-00001 TABLE 1 List of transitions used for quantification within LC-PRM analysis Peptide Modified Precursor Precursor Product Product Fragment Sequence Mz Charge Mz Charge lon DLQAQVVESAK (light) 594.317 2 632.361367 1 y6 DLQAQVVESAK (light) 594.317 2 533.292953 1 y5 DLQAQVVESAK (light) 594.317 2 434.224539 1 y4 DLQAQVVESAK (light) 594.317 2 305.181946 1 y3 DLQAQVVESAK (light) 594.317 2 218.149918 1 y2 DLQAQVVESAK (light) 594.317 2 229.118283 1 b2 DLQAQVVESAK (light) 594.317 2 357.176861 1 b3 DLQAQVVESAK (light) 594.317 2 428.213974 1 b4 DLQAQVVESAK (light) 594.317 2 556.272552 1 b5 DLQAQVVESAK (light) 594.317 2 655.340966 1 b6 DLQAQVVESAK (heavy) 598.3241 2 640.375566 1 y6 DLQAQVVESAK (heavy) 598.3241 2 541.307152 1 y5 DLQAQVVESAK (heavy) 598.3241 2 442.238738 1 y4 DLQAQVVESAK (heavy) 598.3241 2 313.196145 1 y3 DLQAQVVESAK (heavy) 598.3241 2 226.164117 1 y2 DLQAQVVESAK (heavy) 598.3241 2 229.118283 1 b2 DLQAQVVESAK (heavy) 598.3241 2 357.176861 1 b3 DLQAQVVESAK (heavy) 598.3241 2 428.213974 1 b4 DLQAQVVESAK (heavy) 598.3241 2 556.272552 1 b5 DLQAQVVESAK (heavy) 598.3241 2 655.340966 1 b6

Example 2

Quantification of Plasma Proteins Using Vacuum Dried Internal Standard Proteins

Materials and Methods

[0083] 100 isotopically labeled internal standard proteins (in 1 M urea and 1x PBS) targeting 100 human endogenous plasma proteins were mixed in a single container. The mixture was aliquoted to 15 tubes and vacuum dried for 3 hours at 42 C. Sodium deoxycholate (SDC) was diluted in Milli-Q water and added to vacuum dried, isotopically labeled standards so that the final concentrations of SDC, urea and PBS after addition of diluted plasma were 1% SDC, 1 M urea and 1x PBS.

[0084] A pool of plasma from human subjects (3 males, 2 females) was diluted 10 times with 1x PBS. An amount corresponding to 0.5 l of undiluted plasma was added into each of the 15 tubes comprising the vacuum dried mixture of internal standard proteins. Samples were treated in 10 mM DTT at 37 C. for 1 h and 50 mM CAA for 30 minutes at room temperature in the dark. SDC was diluted to a final concentration of 0.25% (w/w) with 1x PBS prior to addition of porcine trypsin (Thermo Scientific) in an enzyme:substrate ratio of 1:50. Digestion was performed at 37 C. and quenched with 0.5% (v/v) trifluoroacetic acid (TFA) after 1, 2, 3, 4 and 16 hours (FIG. 6). Quenched samples were centrifuged at 13,200 rcf for 5 min, and supernatants desalted on 3-layer C18 StageTips prepared in house (Rappsilber et al. (2007), Nat. Protoc. 2:1896-1906). In brief, StageTips were activated with 50 l of 100% ACN and equilibrated with 50 l 0.1% TFA followed by addition of the digested sample corresponding to 15 ug of proteins in raw plasma. The C18 matrix was washed twice with 0.1% TFA and peptides eluted in two steps with 80% ACN, 0.1% TFA. Eluted peptides were vacuum dried at 42 C. Desalted samples were dissolved in Solvent A and an amount corresponding to 4 g protein in undiluted plasma was subjected to LC-MS/MS analysis using data-independent acquisition (DIA).

[0085] Quantification of internal standard proteins usinq LC-DIA: Analysis was performed using an Ultimate 3000 LC online system (Thermo Fisher) connected to a Q Exactive HF MS (Thermo Fisher). First, an amount corresponding to 4 g protein in undiluted plasma was loaded onto a trap column (cat. no. 160438, Thermo Scientific) and washed for 1 min at a flow rate of 15 l/min with Solvent A. Peptides were then separated by a 15 cm analytical column (cat. no. ES806A, Thermo Scientific). A 50 min method with a linear gradient was used for eluting the peptides, ranging from 1% to 32% Solvent B at a flow rate of 3.6 l/min. The analytical column was washed with 99% Solvent B for 30 s followed by two seesaw gradients from 1% to 99% Solvent B. Column was then re-equilibrated for 1 min with 1% Solvent B.

[0086] The MS operated in DIA mode with each cycle comprising of one full MS scan performed at 60,000 resolution (AGC target 3e6, mass range 300-1,200 m/z and injection time 105 ms) followed by 30 DIA MS/MS scans at 30,000 resolution (AGC target 1e6, NCE 26, isolation window 12 m/z, injection time 55 ms), defined by an inclusion list ranging from 350 to 1,000 m/z. Resulting RAW files were loaded into Skyline (v. 20.1.0.76; MacLean et al. (2010), Bioinformatics 26:966-968) and ratios between areas under the curves for heavy peptides from internal protein standards and peptides from endogenous proteins were exported and analyzed.

Results

[0087] A cluster analysis was performed using the exported peptide ratios from the endogenous proteins (light) and isotopically-labelled standard proteins (heavy) at the five time points, resulting in identification of four clusters of peptides (FIG. 7). The two clusters having by far the most members (clusters 2 and 4) exhibit very little variation over time, showing that the digestion efficiency of both isotopically labelled protein standards and the corresponding endogenous proteins in corresponding peptide regions is constant throughout the time course. Cluster 1 shows, for the few members of that cluster, that there is a higher efficiency in digestion of the internal standard protein than of the endogenous protein during the time course. This results in higher amounts of certain peptides from the internal standard protein than of the corresponding peptides from the endogenous protein, as shown by the positive slope of the line before an equilibrium is reached after 16 hours. On the other hand, the few members of cluster 3 exhibit a higher efficiency for the digestion of the endogenous protein than of the isotopically labelled protein standard.

[0088] Most importantly, regardless of where the quantified peptide clusters and regardless of the digestion time, the precision of quantification remains stable and high for all clusters, with median CVs ranging between 4.6% and 6.1% (FIG. 8). This allows for short digestion times and rapid sample preparation protocols with great precision in quantification.

[0089] A set of 100 blood plasma proteins was quantified using a mixture of 100 internal standard proteins that was vacuum dried according to the present disclosure. The proteins were quantified using 292 peptides and cover a plasma concentration span of more than 4 orders of magnitude (10.sup.2-10.sup.2, FIG. 9). The median CV between the technical replicates was 4.6%, demonstrating a great precision in the assay developed.

ITEMIZED LISTING OF EMBODIMENTS

[0090] 1. Solid mixture comprising: [0091] at least one internal standard protein; [0092] at least one chaotropic agent or a derivative or salt thereof; and [0093] optionally a buffer.

[0094] 2. Solid mixture according to item 1, wherein said chaotropic agent is selected from the group consisting of urea, guanidine, thiourea and derivatives and salts thereof.

[0095] 3. Solid mixture according to item 1, wherein said chaotropic agent is urea or a derivative or salt thereof.

[0096] 4. Solid mixture according to item 3, wherein said chaotropic agent is urea.

[0097] 5. Solid mixture according to item 1, wherein said chaotropic agent is guanidine or a derivative or salt thereof.

[0098] 6. Solid mixture according to item 1, wherein said chaotropic agent is thiourea or a derivative or salt thereof.

[0099] 7. Solid mixture according to any one of the preceding items, wherein said at least one internal standard protein remains stable upon storage for at least 1 week, such as at least 2 weeks, such as at least 3 weeks, such as at least 4 weeks, such as at least 5 weeks, such as at least 6 weeks, such as at least 7 weeks, such as at least 8 weeks, such as at least 9 weeks, such as at least weeks, such as at least 3 months, such as at least 6 months, such as at least 1 year, such as at least 2 years.

[0100] 8. Solid mixture according to any one of the preceding items, wherein said at least one internal standard protein remains stable upon storage at a temperature of at least 4 C., such as at least 7 C., such as at least 10 C., such as at least 15 C., such as at least 20 C., such as at least 25 C., such as at least 30 C., such as at least 35 C., such as at least 40 C.

[0101] 9. Solid mixture according to any one of the preceding items, wherein said at least one internal standard protein remains stable upon storage at a temperature of at most 0 C., such as when stored at at most 10 C., such as at most 20 C., such as when stored at at most 50 C., such as when stored at at most 80 C.

[0102] 10. Solid mixture according to any one of the preceding items, wherein said at least one internal standard protein remains stable when subjected to at least 1 freeze-thaw cycle, such as at least 2 freeze-thaw cycles, such as at least 3 freeze-thaw cycles, such as at least 4 freeze-thaw cycles, such as at least 5 freeze-thaw cycles, such as at least 6 freeze-thaw cycles, such as at least 7 freeze-thaw cycles, such as at least 8 freeze-thaw cycles, such as at least 9 freeze-thaw cycles, such as at least 10 freeze-thaw cycles, such as at least freeze-thaw cycles, such as at least 20 freeze-thaw cycles.

[0103] 11. Solid mixture according to any one of items 7-10, wherein said stability is determined by a coefficient of variation.

[0104] 12. Solid mixture according to any one of the preceding items, wherein said at least one internal standard protein is at least 2 standard proteins, such as at least 5 standard proteins, such as at least 10 standard proteins, such as at least 20 standard proteins, such as at least 30 standard proteins, such as at least 40 standard proteins, such as at least 50 standard proteins, such as at least 60 standard proteins, such as at least 70 standard proteins, such as at least 80 standard proteins, such as at least 90 standard proteins, such as at least 100 standard proteins, such as at least 200 standard proteins, such as at least 300 standard proteins, such as at least 400 standard proteins, such as at least 500 standard proteins.

[0105] 13. Solid mixture according to any one of the preceding items, wherein said internal standard protein comprises an isotopic label.

[0106] 14. Solid mixture according to any one of the preceding items, wherein said internal standard protein comprises at least one isotopically labeled amino acid.

[0107] 15. Solid mixture according to any one of items 13-14, wherein said isotopic label is selected from the group consisting of .sup.15N, .sup.13C and .sup.18O.

[0108] 16. Solid mixture according to any one of the preceding items, wherein said internal standard protein is a recombinant protein.

[0109] 17. Solid mixture according to any one of the items 1-15, wherein said internal standard protein is a synthetic protein.

[0110] 18. Solid mixture according to any of the preceding items, wherein said mixture comprising said at least one internal standard protein and at least one chaotropic agent further comprises phosphate.

[0111] 19. Solid mixture according to any one of the preceding items, further comprising a sample suspected to comprise at least one target protein.

[0112] 20. Solid mixture according to item 19, wherein said sample is a bodily fluid sample selected from the group consisting of plasma, serum, blood, cerebrospinal fluid, dry blood spots, saliva and urine.

[0113] 21. Solid mixture according to any one of items 19-20, wherein said sample is solidified.

[0114] 22. Solid mixture according to any one of items 19-21, wherein said internal standard protein comprises a fragment of said target protein.

[0115] 23. Solid mixture according to any one of the preceding items, for use in mass spectrometry.

[0116] 24. Solid mixture according to any one of the preceding items, for use in proteomics.

[0117] 25. Method for preparing a container comprising a solid mixture comprising at least one internal standard protein, the method comprising: [0118] providing a solution comprising said at least one internal standard protein and at least one chaotropic agent selected from the group consisting of urea, guanidine and derivatives and salts thereof, [0119] placing said solution in a container, [0120] removing residual liquid from said solution,
thereby obtaining a container comprising a solid mixture comprising said at least one internal standard protein and said at least one chaotropic agent.

[0121] 26. Method according to item 25, wherein said chaotropic agent is selected from the group consisting of urea, guanidine, thiourea and derivatives and salts thereof.

[0122] 27. Method according to any one of items 25-26, wherein said chaotropic agent is urea or a derivative or salt thereof.

[0123] 28. Method according to any one of items 25-26, wherein said chaotropic agent is guanidine or a derivative or salt thereof.

[0124] 29. Method according to any one of items 25-26, wherein said chaotropic agent is thiourea or a derivative or salt thereof.

[0125] 30. Method according to any one of items 25-29, wherein said chaotropic agent is present in said solution in a concentration of at least 0.5 M, such as at least 1 M, such as at least 2 M, such as at least 3 M, such as at least 4 M, such as at least 5 M, such as at least 6 M, such as at least 7 M, such as at least 8 M.

[0126] 31. Method according to any one of items 25-30, wherein said solution comprising said at least one internal standard protein and at least one chaotropic agent further comprises phosphate.

[0127] 32. Method according to any one of items 25-31, wherein the step of removing residual liquid from said solution comprises removing liquid by means of reduced pressure.

[0128] 33. Method according to item 32, wherein the step of removing liquid by means of reduced pressure is by means of vacuum drying.

[0129] 34. Method according to any one of items 32-33, wherein the step of removing liquid by means of vacuum is at a temperature of 5-60 C., such as at 10-50 C., such as at 15-45 C., such as at 20-45 C., such as at 25-45 C., such as at 30-45 C., such as at 35-45 C., such as 40-45 C., such as at 42 C.

[0130] 35. Method according to item 32, wherein the step of removing liquid by means of reduced pressure is by means of freeze drying.

[0131] 36. Method according to any one of items 25-35, wherein said method provides retained stability of said at least one internal standard protein upon storage for at least 1 week, such as at least 2 weeks, such as at least 3 weeks, such as at least 4 weeks, such as at least 5 weeks, such as at least 6 weeks, such as at least 7 weeks, such as at least 8 weeks, such as at least 9 weeks, such as at least 10 weeks, such as at least 3 months, such as at least 6 months, such as at least 1 year, such as at least 2 years.

[0132] 37. Method according to any one of the items 25-36, wherein said method provides retained stability of said at least one internal standard protein upon storage at a temperature of at least 4 C., such as at least 7 C., such as at least 10 C., such as at least 15 C., such as at least 20 C., such as at least 25 C., such as at least 30 C., such as at least 35 C., such as at least 40 C.

[0133] 38. Method according to any one of items 25-37, wherein said method provides retained stability of said at least one internal standard protein upon storage at a temperature of at most 0 C., such as stored at at most 10 C., such as stored at at most 20 C., such as stored at at most 50 C., such as stored at at most 80 C.

[0134] 39. Method according to any one of items 25-38, wherein said method provides retained stability of said at least one internal standard protein when subjected to at least 1 freeze-thaw cycle, such as at least 2 freeze-thaw cycles, such as at least 3 freeze-thaw cycles, such as at least 4 freeze-thaw cycles, such as at least 5 freeze-thaw cycles, such as at least 6 freeze-thaw cycles, such as at least 7 freeze-thaw cycles, such as at least 8 freeze-thaw cycles, such as at least 9 freeze-thaw cycles, such as at least 10 freeze-thaw cycles, such as at least 15 freeze-thaw cycles, such as at least 20 freeze-thaw cycles, such as at least 50 freeze-thaw cycles.

[0135] 40. Method according to any one of items 37-39, wherein said retained stability is determined by a coefficient of variation.

[0136] 41. Method according to any one of items 25-40, wherein said at least one internal standard protein is at least 2 standard proteins, such as at least 5 standard proteins, such as at least 10 standard proteins, such as at least 20 standard proteins, such as at least 30 standard proteins, such as at least 40 standard proteins, such as at least 50 standard proteins, such as at least 60 standard proteins, such as at least 70 standard proteins, such as at least 80 standard proteins, such as at least 90 standard proteins, such as at least 100 standard proteins, such as at least 200 standard proteins, such as at least 300 standard proteins, such as at least 400 standard proteins, such as at least 500 standard proteins.

[0137] 42. Method according to any one of items 25-41, wherein said internal standard protein comprises an isotopic label.

[0138] 43. Method according to any one of items 25-42, wherein said internal standard protein comprises at least one isotopically labeled amino acid.

[0139] 44. Method according to any one of items 42-43, wherein said isotopic label is selected from the group consisting of .sup.15N, .sup.13C and .sup.18O.

[0140] 45. Method according to any one of items 25-44, wherein said internal standard protein is a recombinant protein.

[0141] 46. Method according to any one of items 25-45, wherein said internal standard protein is a synthetic protein.

[0142] 47. Container comprising a solid mixture according to any one of items 1-18.

[0143] 48. Container comprising a solid mixture according to any one of items 19-22.

[0144] 49. Container obtainable by a method according to any one of items 25-46.

[0145] 50. Container according to any one of items 47-49, which is selected from the group consisting of a microtiter plate, a vial, a collection tube, a bottle, a pre-coated filter paper, a blood tube, a Whatman paper, a DBS collection device, a dried plasma spot device, a dried serum spot device and a culturing plate.

[0146] 51. Container according to any one of items 47-50, for use in mass spectrometry.

[0147] 52. Container according to any one of items 47-51, for use in proteomics.

[0148] 53. Method for determining the amount of a target protein present in a sample, the method comprising: [0149] providing a container according to item 47; [0150] adding a sample suspected of comprising at least one target protein to said mixture, thereby preparing a test sample, [0151] subjecting said test sample to analysis, [0152] using the results of the analysis to determine the amount of said at least one target protein in said sample by comparison with said internal standard protein.

[0153] 54. Method for determining the amount of a target protein present in a sample, the method comprising: [0154] providing a container according to item 48; [0155] subjecting said sample to analysis, [0156] using the results of the analysis to determine the amount of said at least one target protein in said sample by comparison with said internal standard protein.

[0157] 55. Method for determining the amount of a target protein present in a sample, the method comprising: [0158] providing a container according to item 49; [0159] unless already present, adding a sample suspected of comprising at least one target protein to said solid mixture, thereby preparing a test sample, [0160] subjecting said test sample to analysis, [0161] using the results of the analysis to determine the amount of said at least one target protein in said sample by comparison with said internal standard protein.

[0162] 56. Method according to any one of items 51-54, wherein said internal standard protein comprises a fragment of said target protein.

[0163] 57. Method according to any one of items 51-56, wherein said analysis is performed using mass spectrometry.

[0164] 58. Method according to any one of items 51-57, wherein the method further comprises removing residual liquid from said sample.

[0165] 59. Method according to any one of items 51-58, wherein the method further comprises a step of long-term storage of said sample preceding the steps of subjecting said sample to analysis and determining the amount of said at least one target protein in said sample by comparison with said standard protein.

[0166] 60. Method according to item 59, wherein said long-term storage is for at least 1 week, such as at least 2 weeks, such as at least 3 weeks, such as at least 4 weeks, such as at least 5 weeks, such as at least 6 weeks, such as at least 7 weeks, such as at least 8 weeks, such as at least 9 weeks, such as at least 10 weeks, such as at least 3 months, such as at least 6 months, such as at least 1 year, such as at least 2 years.

[0167] 61. Method according to any one of items 53-60, wherein said sample is a bodily fluid sample selected from the group consisting of plasma, serum, blood, cerebrospinal fluid, dry blood spots, saliva and urine.

[0168] 62. Kit for carrying out the method according to any one of items 53-61, the kit comprising: [0169] a container according to any one of items 47-52, and [0170] instructions for carrying out the method.