SAMPLE PREPARATION FOR PROTEOMIC INVESTIGATIONS

Abstract

A method for disrupting a sample of biological material for proteome analysis by a mass-spectrometry method is provided, involving admixing the sample with a certain volume of an organic acid, incubating said sample for a certain period for lysis and then neutralizing said sample.

Claims

1. A method for disrupting a sample of biological material for proteome analysis by a mass-spectrometry method, comprising the steps of: providing the sample in a reaction vessel, adding a certain amount of an organic acid to the sample, incubating the preparation, adding a neutralizing solution to the preparation.

2. The method as claimed in claim 1, wherein the biological material encompasses samples of human, animal or plant origin, especially tissues, cells, body fluids, blood and blood products, swabs as well as feces.

3. The method as claimed in claim 1, wherein the method comprises the additional step of electromagnetic irradiation or of heating of the sample preparation.

4. The method as claimed in claim 3, wherein the irradiation carried out is a microwave irradiation or the sample is heated to over 40 C.

5. The method as claimed in claim 1, wherein the organic acid used is a carboxylic acid.

6. The method as claimed in claim 1, wherein the organic acid used is a haloacid.

7. The method as claimed in claim 1, wherein the organic acid used is trifluoroacetic acid and the derivatives thereof.

8. The method as claimed in claim 1, comprising the further steps of: adding an alkylation solution, adding a reduction solution, determining the protein concentration, diluting the sample with water, enzymatically or chemically cleaving the proteins, purifying the peptides generated.

9. (canceled)

10. (canceled)

11. A method for lysing a biological material, comprising: adding trifluoroacetic acid to the biological material; and lysing the biological material.

12. A method for conducting mass spectrometry on a biological material, comprising: obtaining the biological material according to the method of claim 1; and conducting mass spectrometry on the obtained biological material.

Description

[0031] The invention will be more particularly elucidated with reference to the figures, where:

[0032] FIG. 1 shows a flow chart of one embodiment of the method according to the invention.

[0033] FIG. 2 shows a comparison of the lysis of samples.

[0034] FIG. 3 shows results of a proteome analysis of human cells from the described method in comparison with a reference method (triplicates).

[0035] In one embodiment of the method according to the invention as per the depiction in FIG. 1, a sample of a biological material is provided in a first step S1. The biological material can be a tissue sample, for example a blood sample, or a suspension or a pellet of cells and also solid tissue structures.

[0036] In a second step S2-1, pure trifluoroacetic acid is added in excess to the biological material, ideally in a volume ratio between 1:1-1:10 (sample to TFA). The preparation is mixed, for example by means of a vortexer, and incubated at room temperature for 1-10 min. In the course of this, the sample material is lysed. In addition to the chronological sequence, the course of lysis is followed visually; when the solution becomes clear, the sample material has been lysed (FIG. 2). If the protein concentration is too high for extraction with the specified amount of acid, an appropriate further volume of acid is added, for example 100 l. In an optional step S2-2, which supports the disruption of difficult-to-lyse materials, the material is incubated in a microwave at an output of 800 W for 10 s.

[0037] To illustrate the action of acid on the samples, FIG. 2 depicts various sample types, before and after lysis in each case. Depicted from left to right are: a suspension of HeLa cells (human), two skin samples (chicken) and one liver sample (chicken). Depicted in each case are the starting material as well as the samples after lysis with TFA and after performance of neutralization. Whereas the HeLa cells and the liver tissue are completely lysed by sole addition of acid, undissolved tissue constituents become apparent in one of the skin samples (second sample from the left; see the arrows) after addition of TFA and after neutralization. Said constituents can be dissolved when the sample is irradiated with microwaves (Skin* sample; see the third sample from the left).

[0038] After the solution becomes clear, the lysed material preparation is neutralized with an aqueous 2 M TRIS solution in a third step S3. In this connection, one volume of the sample is neutralized by adding about 8-15 times the volume of a 2 M TRIS solution, i.e., for a 100 l lysis preparation, 1000 l of 2 M TRIS solution are added.

[0039] In any case, the pH of the solution is checked in step S3. What is striven for in this connection is a pH between 7 and 9, ideally between 8 and 8.5. To reach the striven-for pH, readjustment is carried out if necessary by adding 2 M TRIS or a diluted trifluoroacetic acid solution until the optimum pH is reached.

[0040] After the neutralization, the solution is admixed with a reduction and alkylation solution in a fourth step S4. To this end, a freshly prepared solution of 100 mM TCEP (tris(2-carboxyethyl)phosphine) and 400 mM CAA (chloroacetamide) in water is used, of which 10% of the sample volume are added to the sample. The samples are incubated in a thermal mixer at 95 C. for a period of at least 3-10 min. After this step, the samples can be frozen and can be stored at temperatures 20 C. for a relatively long period. Step S4 can be omitted if peptides containing no cysteines are being analyzed. It is also possible to use alternative reduction and alkylation reagents, such as, for example, DTT (dithiothreitol) or IAA (iodoacetamide), and in this case the incubation conditions must be adjusted accordingly.

[0041] In a fifth step S5, the method is continued by cleaving the proteins of the sample enzymatically or chemically into peptides. To this end, in the case of an enzymatic digestion, the protein concentration of the sample is determined in a manner known to a person skilled in the art, and trypsin or another suitable enzyme is then added to the sample in a defined mass ratio, this being within the range from 500:1 to 5:1 for trypsin (protein:trypsin). The sample can be diluted with water according to the dependence of the activity of the enzyme used on the buffer concentration of the lysate. The digest preparation is then incubated at a temperature optimum for the enzyme (37 C. for trypsin) for an appropriate length of time (1-24 h for trypsin).

[0042] In a sixth step S6, the peptides are purified, for example by a solid-phase extraction on C18 material.

[0043] The purified peptides can then be analyzed by means of liquid chromatography coupled with mass spectrometry (LC-MS) or using comparable methods such as capillary electrophoresis coupled with mass spectrometry. FIG. 3 depicts a comparison of the results of a proteome investigation of human cells with an alternative sample preparation method. For this purpose, the coefficient of variation (CV) was shown against the signal intensity for peptides and for the resulting proteins in a scatter plot. The cloud of points was overlaid with a heat map. The number of quantified peptides and proteins as well as the mean values of the CVs are likewise indicated. On the left-hand side, the graphs depict results from a sample which was disrupted using the method according to the invention as per FIG. 1, and the graphs on the right-hand side show results from a sample disrupted using conventional methods. It can be seen that more peptides and, derived therefrom, more proteins can be captured at a lower signal variance using the method according to the invention than using a conventional method.