Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/04—Preparation or injection of sample to be analysed
  • G01N30/06—Preparation
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/62—Detectors specially adapted therefor
  • G01N30/72—Mass spectrometers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5067—Liver cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5097—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving plant cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/04—Preparation or injection of sample to be analysed
  • G01N30/06—Preparation
  • G01N2030/067—Preparation by reaction, e.g. derivatising the sample
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/24—Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Definitions

• the invention relates to a method for processing biological material for proteomic investigations, the material being lysed using an organic acid.
• proteome comprises the entirety of all proteins expressed in a cell or a living being at a certain point in time under certain conditions.
• proteomics the central technology of which is currently liquid chromatography coupled with mass spectrometry (LC-MS). Due to the immense importance of proteins for the phenotype of living things, proteomic techniques are increasingly used in medicine and the life sciences, among other things to identify biomarkers for diseases or to adapt therapy methods to patients in the future.
• proteomics essentially comprise three essential steps, (i) lysis / extraction, (ii) digestion and (iii) peptide purification.
• the sample preparation can be extended by additional steps, such as purification or labeling reactions.
• the current state of the art is based on the extraction of proteins using detergents (e.g. sodium lauryl sulfate (SDS), deoxycholic acid (SDC)) and / or chaotropic substances (e.g. urea, guanidine hydrochloride (GnHCI)), often supported by physical energy input (ultrasound, Pressure, friction, grinding, heat) to achieve an effective lysis of the sample material.
• detergents e.g. sodium lauryl sulfate (SDS), deoxycholic acid (SDC)
• chaotropic substances e.g. urea, guanidine hydrochloride (GnHCI)
• a first aspect of the invention relates to a method for the digestion of a sample of biological material for proteome analysis by a mass spectrometric method, which comprises the following work steps:
• the method according to the invention is advantageous because fewer work steps are required compared to conventional methods. This makes it more economical in terms of time and workload than conventional methods. Furthermore, with sample material digested according to the invention, a greater intensity of the signals with a lower variance is advantageously obtained compared to conventional methods (FIG. 3). Another advantage is the high reproducibility of the process, which is particularly important with regard to quantitative analyzes. In addition, proteins in such solutions with a high salt concentration are very stable, which considerably facilitates transport, storage and archiving of the samples.
• the method can advantageously be used for any type of biological material that contains proteins that are to be proteomically analyzed.
• the biological material preferably comprises samples of human, animal or vegetable origin, in particular tissue, cells, body fluids, blood and blood products, smears and faeces.
• the biological material is provided in a reaction vessel made of a suitable material and in a suitable size.
• the amount of organic acid to be added is at the discretion of those skilled in the art. For a known amount of biological material, a standardized amount of acid is ideally added. Ideally, a sample-to-acid ratio is set so that the sample material dissolves completely.
• the sample material can be easily incubated with the acid for up to 60 min at room temperature without affecting the quality of the proteins.
• the lysis can be stopped as soon as the suspension becomes clear, which is the case for most samples after 2-10 minutes.
• the sample is neutralized in order to prepare the proteins for digestion.
• a weak base is used for neutralization, for example an aqueous 2M TRIS solution (tris (hydroxymethyl) aminomethane), the volume of the TRIS solution being supposed to be about 7-15 times the sample volume.
• the aim is to achieve a pH value between 7 and 9, ideally between 8 and 8.5.
• the method preferably comprises an additional step of electromagnetic radiation directly before the neutralization.
• This step is particularly advantageous for samples that are difficult to digest, for example samples with a high collagen content.
• a thermal treatment at temperatures> 40 ° C shows comparable effects.
• the sample is particularly preferably irradiated by microwaves.
• the sample is exposed to a microwave power of 800 W for a period of 5-30 s, especially for 10 s. It is advantageous to provide the reaction vessel in which the sample is incubated with an additional covering, e.g. another plastic container. It has been found that the microwave treatment is advantageous for effective lysis for any type of biological material, but especially for material that is difficult to lyse.
• a carboxylic acid is preferably used as the organic acid in the process.
• a halocarboxylic acid is more preferably used in the process.
• Trifluoroacetic acid (TFA) and its derivatives are particularly preferably used as organic acid in the process.
• TFA has been shown to be particularly effective in lysing tissues and cells. The use of TFA is also advantageous because the analysis of the proteins is not affected by the action of the TFA.
• the method preferably comprises the further steps:
• a second aspect of the invention relates to the use of trifluoroacetic acid for the lysis of biological material.
• TFA is suitable for every biological material mentioned above.
• a third aspect of the invention relates to the use of a sample of biological material prepared by the method according to the invention in a mass spectrometric method.
• Figure 1 is a flow diagram of an embodiment of the method according to the invention.
• FIG. 2 shows a comparative illustration of the lysis of samples.
• Figure 3 Results of a proteome analysis of human cells of the method described in comparison with a reference method (triplicate).
• a sample of a biological material is provided in a first step S1.
• the biological material can be a tissue sample, e.g. a blood sample, or a suspension or a pellet of cells, as well as solid tissue structures.
• a second step S2-1 pure trifluoroacetic acid is added in excess to the biological material, ideally in one Volume ratio between 1: 1-1: 10 (sample to TFA).
• the mixture is mixed, for example using a vortex device, and incubated at room temperature for 1-10 min.
• the sample material is lysed.
• the course of lysis is monitored visually; when the solution becomes clear, the sample material has been lysed (Fig. 2). If the protein concentration is too high to be extracted with the specified amount of acid, an appropriate additional volume of acid is added, for example 100 ml.
• an optional step S2-2 which supports the digestion of material that is difficult to lyse, the material is incubated for 10 s at a power of 800 W in a microwave oven.
• sample types are shown in FIG. 2, each before and after the lysis. From left to right are shown: a suspension of HeLa cells (human), two skin samples (chicken) and a liver sample (chicken). The starting material and the samples after lysis with TFA or after neutralization are shown. While the HeLa cells as well as the liver tissue are completely lysed by the addition of acid alone, tissue components which have not been dissolved appear after addition of TFA or after neutralization in one of the skin samples (second sample from left, see arrows). These components can be dissolved if the sample is irradiated with microwaves (sample skin * , see third sample from left).
• the batch of the lysed material is neutralized in a third step S3 with an aqueous 2M TRIS solution.
• an aqueous 2M TRIS solution To neutralize a volume of the sample, approximately 8-15 times the volume of a 2M TRIS solution is added, i.e. for 100 mI lysis approach, 1000 mI of 2M TRIS solution are added.
• the pH of the solution is checked in step S3.
• the aim is to achieve a pH value between 7 and 9, ideally between 8 and 8.5.
• 2M TRIS or a dilute trifluoroacetic acid solution is added until the optimal pH is reached.
• the solution is mixed with a reduction and alkylation solution in a fourth step S4.
• a freshly prepared solution of 100 mM TCEP (tris (2-carboxyethyl) phosphine) and 400 mM CAA (chloroacetamide) in water is used for this, from which 10% of the sample volume is added to the sample.
• the samples are incubated for at least 3-10 min at 95 ° C in a thermomixer.
• Step S4 can be omitted if peptides are analyzed which do not contain cysteines.
• Alternative reduction and alkylation reagents such as DTT (dithiothreitol) or IAA (iodoacetamide) can also be used, the incubation conditions having to be adapted accordingly.
• a fifth step S5 the method is continued by enzymatically or chemically cleaving the proteins of the sample into peptides.
• the protein concentration of the sample is determined in a manner known to the person skilled in the art, and then trypsin or another suitable enzyme in a defined mass ratio, for trypsin in the range between 500: 1 to 5: 1 (protein: trypsin), given for rehearsal.
• the sample can be diluted with water depending on the activity of the enzyme used and the buffer concentration of the lysate.
• the digestion is then incubated at a temperature that is optimal for the enzyme (37 ° C. for trypsin) for a suitable time (1-24 h for trypsin).
• a sixth step S6 the peptides are purified, e.g. by solid phase extraction on C18 material.
• the purified peptides can then be analyzed using liquid chromatography coupled with mass spectrometry (LC-MS) or with comparable methods such as capillary electrophoresis coupled with mass spectrometry.
• LC-MS liquid chromatography coupled with mass spectrometry
• comparable methods such as capillary electrophoresis coupled with mass spectrometry.
• 3 is a comparison of the results of a proteome examination of human cells with an alternative Sample preparation method shown.
• CV coefficient of variance
• the point cloud was overlaid with a HeatMap representation.
• the number of peptides and proteins quantified and the mean values of the CVs are also given.
• results are shown which were disrupted using the method according to the invention according to FIG.

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• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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• 2018
  • 2018-06-12 DE DE102018114028.2A patent/DE102018114028A1/de not_active Withdrawn
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