# US 11,307,122 B2 · Preparation of biological cells on mass spectrometric sample supports for desorbing ionization > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,307,122 B2 - Document ID: US11307122B2 - Status: Granted - Kind: B2 - Application number: 15/968,428 - Filed: 2018-05-01 - Published: 2018-11-15 - Granted: 2022-04-19 - Earliest claimed: 2017-05-15 - Applicants: Bruker Daltonik GmbH - Inventors: Katrin Sparbier; Beatrix Wegemann - Web page: https://patentgrep.com/patent/US11307122B2 ## CPC - G01N 1/4044: {by chemical techniques; Digestion; Chemical decomposition} - C12Q 1/045: {Culture media therefor} - C12Q 1/18: Testing for antimicrobial activity of a material - G01N 1/28: Preparing specimens for investigation {including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q}(mounting specimens on microscopic slides G02B21/34; means for supporting the objects or the materials to be analysed in electron microscopes H01J37/20 {; laboratory gas handling apparatus B01L5/00}) - G01N 33/6851: {Methods of protein analysis involving laser desorption ionisation mass spectrometry} - G01N 1/4055: {by solubility techniques} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The invention relates to the preparation of biological cells for the mass spectrometric analysis of cellular properties such as taxonomic classification, antibiotic resistances, response to drugs or other active substances, and others. The cells can be prokaryotic or eukaryotic microorganisms which have particularly been cultivated directly on a mass spectrometric sample support, or eukaryotic cells from tissues or cell cultures. The invention proposes that the cells are not disrupted by adding matrix solution for a subsequent ionization by matrix-assisted laser desorption (MALDI), but that they are disrupted in a separate treatment step using acids and/or solvents on the sample support itself. Surprisingly, the cell proteins released then adhere to the sample support so that they can be carefully washed with buffer solution to remove salts and other soluble impurities which can stem from earlier treatment steps, for example from nutrient solution. ## Claims ### Claim 1 A method for the preparation of proteins from samples of biological cells for the mass spectrometric determination of cellular properties, comprising the steps: cultivating the biological cells in nutrient liquid directly on a sample spot of a mass spectrometric sample support, disrupting the cultivated cells on the sample spot using solvents and/or organic acids, whereby cell proteins separate from complexes, migrate out of the disrupted cells and are adhesively bonded to the sample spot, washing the cell proteins on the sample spot with buffer solution, and preparing the washed cell proteins on the sample spot for a subsequent desorbing ionization. ### Claim 2 (depends on 1) The method according to claim 1, wherein the washing step is conducted with a static buffer solution. ### Claim 3 (depends on 2) The method according to claim 2, wherein, for the washing step, several microliters of an aqueous solution are applied to the sample spot, remain there for a predetermined period of time and are then removed. ### Claim 4 (depends on 1) The method according to claim 1, wherein the preparation for the desorbing ionization comprises the addition and crystallization of matrix solution on the sample spot for a subsequent ionization by matrix-assisted laser desorption. ### Claim 5 (depends on 1) The method according to claim 1, wherein the mass spectrometric sample support comprises a stainless steel plate, a plate with hydrophilic anchors in a hydrophobic environment, or a plate made of coated ceramic material. ### Claim 6 (depends on 1) The method according to claim 1, wherein the biological cells comprise prokaryotic microorganisms, eukaryotic microorganisms or eukaryotic cells from tissues. ### Claim 7 (depends on 6) The method according to claim 6, wherein one of said cellular properties of the biological cells to be determined is their taxonomic classification, resistance to an antimicrobial substance, or response to drugs or other active substances. ### Claim 8 (depends on 7) The method according to claim 7, wherein the antimicrobial substance is an antibiotic or antimycotic. ### Claim 9 (depends on 7) The method according to claim 7, wherein the biological cells are cultivated on different sample spots of the sample support in nutrient liquid with and without the addition of an antimicrobial substance, and their resistance to the antimicrobial substance is determined by determining a difference in growth between a first cultivation in the presence of the antimicrobial substance and a second cultivation in the absence of the antimicrobial substance, by comparing mass spectrometric signals of the cell proteins from the first cultivation and mass spectrometric signals of the cell proteins from the second cultivation. ### Claim 10 (depends on 7) The method according to claim 7, wherein the biological cells are cultivated on different sample spots of the sample support in nutrient liquid with and without the addition of an antimicrobial substance, and their resistance to the antimicrobial substance is determined by further growth in the presence of the antimicrobial substance, which further growth is determined by comparing mass spectrometric signals of the cell proteins with mass spectrometric signals of a reference substance added in a dosed quantity. ### Claim 11 (depends on 7) The method according to claim 7, wherein a minimum inhibitory concentration of the antimicrobial substance is determined for the biological cells. ### Claim 12 (depends on 1) The method according to claim 1, wherein cultivating the biological cells comprises cultivating the biological cells in the presence of chemicals, and wherein the biological cells are mass spectrometrically investigated as to their response to the chemicals. ### Claim 13 (depends on 1) The method according to claim 1, wherein the cellular properties are determined on the basis of mass spectrometric protein signals in a mass range above m/z 3,000. ### Claim 14 (depends on 13) The method according to claim 13, wherein said mass range extends between m/z 3,000 and m/z 15,000. ### Claim 15 (depends on 1) The method according to claim 1, wherein the solvents and organic acids used for disrupting the cells are matrix-free. ### Claim 16 (depends on 1) The method according to claim 1, wherein the cultivated biological cells are unpurified. ### Claim 17 (depends on 1) The method according to claim 1, wherein the buffer solution comprises pure deionized or acidified water. ### Claim 18 (depends on 1) The method according to claim 1, wherein cultivating the biological cells comprises incubating the mass spectrometric sample support in a chamber which provides constant conditions. ### Claim 19 (depends on 1) The method according to claim 1, wherein soluble impurities are removed by said washing. ### Claim 20 (depends on 19) The method according to claim 19, wherein the soluble impurities encompass salts, detergents, and buffer materials. ### Claim 21 (depends on 1) The method according to claim 1, wherein the nutrient liquid used for cultivating the biological cells comprises meat extract and hydrolyzed casein.