Ready-to-use calibration compositions and methods for calibrating mass spectrometers

Abstract

The present invention relates to a ready-to-use calibration composition for mass spectrometers, comprising all E. coli suspended in an alcohol and / or acetonitrile-based liquid, which has sanitizing properties, is volatile, and does not denature bacterial proteins.

Description

[Technical Field]

[0001] The present invention relates to the field of mass spectrometry, which finds various applications in the fields of biomedical research, medicine, diagnostics, and biotechnology. More precisely, the present invention relates to calibration compositions and methods for calibrating mass spectrometers using a matrix-assisted desorption / ionization technique known as MALDI. In particular, the present invention is suitable for the matrix-assisted desorption / ionization time-of-flight technique known as MALDI-TOF. The object of the calibration compositions and calibration methods according to the present invention is to ensure reliable calibration of the spectrometer used, thereby enabling sufficiently accurate measurements of the mass of ions generated from the sample to be analyzed.

[0002] The present invention also relates to the use of a calibration composition according to the present invention as an external standard for calibrating a mass spectrometer, and to a method for characterizing a sample containing at least one microorganism.

[0003] The analysis of microorganisms, more generally by mass spectrometry, particularly by MALDI or MALDI-TOF, of samples that may contain microorganisms has been used for many years to rapidly identify microorganisms and to determine their resistance to specific antimicrobial agents, as exemplified, for example, in the patent application WO2016 / 0166580 in the name of the present applicant. Microbial identification is performed based on the mass spectrum of the most abundant protein in the microorganism, and by comparison with reference data, the family, genus, and usually species of the microorganism can be identified in particular.

[0004] A routine protocol involves depositing the sample to be analyzed, which may contain microorganisms, onto a MALDI analysis plate, adding a matrix suitable for the MALDI method, obtaining a mass spectrum using a MALDI-TOF mass spectrometer, and identifying the species by comparison with reference data stored in a database. The MALDI-TOF method is also used to detect microbial resistance to antibiotics, and in particular to identify phenotypes involved in the hydrolysis of β-lactam antibiotics by the secretion of β-lactamase enzymes, especially carbapenemase enzymes.

[0005] Various mass spectrometers suitable for such characterization are sold, particularly by the applicant (specifically under the reference names VITEK® MS and VITEK® MS Prime), and also by Bruker Daltonics and Autobio. These spectrometers are of the MALDI-TOF type and consist of a laser ionization source and a time-of-flight mass spectrometer. They are intended to operate with an analytical plate, also called a MALDI plate or MALDI target, on which the sample to be analyzed is deposited in combination with a matrix suitable for the MALDI method, and the matrix can be deposited simultaneously with or after the sample.

[0006] Next, the analysis plate is introduced into the analysis chamber of the spectrometer, which is, for example, 10 -5 mbar(10 -3 Pressure less than Pa, typically 10 -6 ~10 -9 mbar(10 -4 ~10 -7A relatively high level of vacuum is created at a pressure in the range of Pa. Under these vacuum conditions, the sample placed in the MALDI matrix is ​​subjected to soft laser ionization. The matrix then absorbs photon energy, and the recovery of this energy leads to the sublimation of the matrix, the desorption of molecules present in the sample, and the emergence of a state of matter called plasma. Within this plasma, charge exchange occurs between molecules from the matrix and molecules from the sample, particularly those from microorganisms. For example, protons can be detached from the matrix and transferred to proteins, peptides, and organic compounds present in the sample.

[0007] This process allows for the gentle ionization of existing molecules without causing their destruction. Thus, the sample releases ions of various sizes. In the MALDI-TOF analyzer, the generated ions are accelerated by an electric field and fly freely within a reduced-pressure tube called a flight tube. This allows for the separation of ions, as the smallest ions "move" faster than the larger ones. The detector is located at the end of the flight tube. In this way, a mass spectrum is obtained, where the signal intensity corresponding to the number of ionized molecules with a given mass-to-charge ratio [m / z] is expressed as a function of the time-of-flight (TOF) of the molecules colliding with the detector. The time-of-flight of the ions is used to calculate the mass-to-charge ratio [m / z], which is expressed in Thomson (Th) terms.

[0008] Calibration of a mass spectrometer improves the reliability of the measurements performed and thus enhances the usability of the generated mass spectrum, particularly for the identification of microorganisms in a sample. Calibration involves adjusting the obtained experimental mass-to-charge ratio value to a "calibrated" value, which will appear in the generated mass spectrum and be used to characterize the sample to be analyzed.

[0009] Such calibration can be performed using an internal calibrator incorporated into the sample, or an external calibrator called an external standard deposited in at least one deposition zone of the MALDI analysis plate.

[0010] As shown in Figure 1, MALDI analysis plate I is typically, - A series of deposition zones 1 (also called spots) called analysis zones for the deposition of the sample to be characterized; after drying, these zones 1 form what are called characterization zones, and - One or more deposition zones 2 called reference zones for the deposition of external standards; after drying, these zones 2 form what are called control zones. Includes; These zones are well-shaped and are usually circular.

[0011] MALDI plates are generally manufactured from materials such as stainless steel, gold, silicon, metal-coated plastic polymers, or plastic polymers reinforced with conductive agents (such as carbon black), or coated glass. They typically have a textured surface to facilitate the deposition of the sample to be analyzed and external standards.

[0012] To facilitate subsequent ionization, the plate surface is generally conductive, at least in the analysis zone and the reference deposition zone. For example, such an analysis plate is formed from a polymer such as polypropylene, which is covered with a layer of stainless steel. The polymer may contain conductive materials such as carbon black. Various MALDI plates are commercially available, such as the VITEK® MS plate (disposable) from bioMerieux and the MALDI Biotarget plate (reusable) from Bruker Daltonics. Such a plate typically contains 48 to 96 analysis zones 1 and at least one, two, or three reference zones 2, the size of which may differ from that of the analysis zones 1.

[0013] Currently, for the calibration of the instrument, the applicant recommends using freshly cultured Escherichia coli (E. coli) strain ATCC 8739 as an external standard, as described in the VITEK® MS user manual. The bacteria are cultured on agar at 37°C for 18–24 hours. A certain amount of bacteria is then collected and deposited in the reference zone on a MALDI plate. This calibration method has the inconvenience of requiring the availability of freshly cultured bacteria.

[0014] Furthermore, bacteria were extracted from the agar plate and measured on a MALDI analysis plate for several millimeters. 2 (especially 5-15mm) 2 Depositing them in a dedicated zone and forming a thin, homogeneous layer of cells is a particularly delicate manual process. The quality of the results can vary greatly depending on the operator and the procedure. However, if the external standard deposits are of poor quality, the identification of microorganisms on a MALDI analysis plate containing numerous spots (typically 16-96 analysis spots) for depositing the sample of interest may fail. In such cases, the operator is forced to completely redo the plate preparation.

[0015] Other types of external standards that enable the calibration of mass spectrometers have been proposed in the prior art. For example, BTS (Bacterial Test Standard), sold by Bruker, is an E. coli extract concentrated with two high molecular weight proteins. BTS was developed to function as a quality control for Bruker's MALDI-TOF Biotyper spectrometer. The BTS datasheet shows that its specific composition covers the entire mass range of proteins used by the Biotyper for accurate identification of microorganisms, particularly the range from 3.6 to 17 kDa. Using BTS, the Biotyper performs automated quality control before each identification cycle. The quality control process includes calibration of the mass spectrometer, verification of laser adjustments, and evaluation of spectral quality. The system's performance is ultimately confirmed by the identification of E. coli, which must meet minimum performance standards. Before use, one dose of BTS is placed in approximately 50 μL of solvent consisting of 50% v / v (volume relative to the total volume of solvent) acetonitrile, 47.5% v / v water, and 2.5% v / v trifluoroacetic acid. The mixture is incubated at room temperature for 5 minutes, and various operations such as mixing by pipetting and centrifugation are recommended by the supplier Bruker in document reference number 8290190, September 2017, "IVD Bacterial Test Standard". The resulting 5 μL aliquots can then be stored in a stoppered tube, frozen at -18°C or below, and subsequently used to form deposits in four different deposition zones of a MALDI plate.

[0016] Patent CN104931572B can also be mentioned. This patent, like Bruker's BTS, proposes a bacterial extract type calibration composition, but with the advantage for the user that it is ready to use. This bacterial extract / lysate is prepared according to the following method, according to the available machine translation: (1) Select strains ATCC 8739, MG 1655, and JM 109 of Escherichia coli, culture them until they reach the logarithmic growth stage, and mix them according to a mass ratio of 3-8:1-5:1-3; (2) Protein washing solution A, which is deionized water or ultrapure water, is added to the strain mixture in a mass / volume ratio of 5-10 mg:0.1-0.5 mL, and then protein washing solution B, which is anhydrous ethanol, is added in a volume ratio of 5-2:1; after mixing and centrifugation, the supernatant is removed; (3) Add protein solubility solution A to the precipitate obtained in step (2) in a mass / volume ratio of (5-10 mg):(0.025-0.125 mL), mix well, then add the same amount of protein solubility solution B as protein solubility solution A, centrifuge, and use the supernatant as the calibration composition; protein solubility solution A is an HPLC-grade 50%-80% formic acid solution, and protein solubility solution B is an HPLC-grade acetonitrile.

[0017] Therefore, it is clear that this calibration composition is complex to manufacture, requiring multiple delicate preparation steps.

[0018] Taking into consideration the prior art calibration compositions and the defects pointed out, the present invention proposes to provide a novel calibration composition that combines the following advantages: - It is easy to use and handle, and in particular, it does not require special experience or skill from the user in terms of the operation of collecting an appropriate amount of bacteria and depositing it in a thin, homogeneous layer. - A ready-to-use preparation with a sufficiently long shelf life. - Simple and inexpensive industrial production.

[0019] In this context, the present invention proposes a calibration composition for a mass spectrometer comprising all E. coli suspended in an alcohol and / or acetonitrile-based liquid.

[0020] For the proposed uses, the inventors have observed and demonstrated that alcohols of the "unsaturated aliphatic alcohol" type (such as methanol, ethanol, propanol, and butanol) and acetonitrile are particularly advantageous due to their good volatility, sterilizing action that is non-denaturing to bacteria, and chemical / reaction neutrality towards proteins. As a result, the calibration composition according to the invention is formulated in the form of a liquid having an essentially volatile sterilizing action, thereby enabling good and sustainable preservation of bacteria and their proteins.

[0021] For the purpose of simplifying the text, herein, the term "alcohol" refers to unsaturated aliphatic alcohols, particularly unsaturated C1-C4 aliphatic alcohols, preferably ethanol and isopropanol.

[0022] Thus, the calibration composition according to the invention contains all the bacteria preserved in this state until the moment the calibration operation is initiated. By being maintained in such an environment, bacterial proteins, particularly the proteins selected as references for calibration, can enjoy protection from biological and / or chemical degradation brought about by the structure of the bacteria themselves remaining intact, as well as by alcohol and / or acetonitrile. The alcohol and / or acetonitrile of the calibration composition according to the invention exert a sterilizing action on the entire composition while themselves exhibiting advantageous chemical / reaction neutrality with respect to proteins.

[0023] In addition to ensuring good and persistent preservation of bacteria and their proteins, such liquid and volatile formulations confer the advantages that they can be used directly as such in the calibration compositions according to the invention and that they greatly facilitate the calibration operation of spectrometers. When using the calibration compositions according to the invention, it is sufficient for an operator to take a predetermined volume (which corresponds to a predetermined amount of cells) and deposit it on an analysis plate. For this purpose, it is possible to use a pipette or other suitable instrument. The high volatility of the alcohol and / or acetonitrile constituting the liquid phase of the calibration composition according to the invention enables the rapid evaporation of the liquid phase, leaving only a thin and homogeneous layer of cells on the deposition zone. Once the matrix is added and the analysis plate is loaded into the spectrometer, data acquisition can be started. Similarly, it is also possible to mix the calibration composition with the matrix, deposit a certain volume of this mixture on the analysis plate, and load it into the spectrometer once the mixture has dried sufficiently.

[0024] Advantageously, the liquid phase of the calibration composition according to the invention is alcohol-based. According to a preferred embodiment, it is an aqueous liquid having an alcohol content of at least 20% v / v, typically 70% v / v.

[0025] Advantageously, according to the invention, the alcohol comprised in the calibration composition according to the invention is ethanol and / or isopropanol. According to a particularly preferred embodiment, the alcohol-based liquid of the calibration composition according to the invention consists of an aqueous water / ethanol mixture having an ethanol content of 70%.

[0026] According to another preferred embodiment, the liquid phase of the calibration composition according to the invention is acetonitrile-based, i.e., a 100% v / v acetonitrile solution or a water / acetonitrile mixture having an acetonitrile content of at least 20% v / v, typically 70% v / v.

[0027] Advantageously, to ensure the simplest possible composition, the Escherichia coli calibration composition according to the present invention is obtained from a single strain. In particular, these bacteria are obtained from the BL21 strain or the ATCC 8739 strain. The BL21 Escherichia coli strain was described in 1986 (Studier FW, Moffatt BA., J. Mol Biol. 1986 May 5;189(1):113-30. “Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes”), and its entirety was characterized by sequencing in 2015 (Jeong H., Ju Kim H., Jun Leeb S., Genome Announc. 2015 Mar 19;3(2).e00134-15. Complete Genome Sequence of Escherichia coli Strain BL21). This strain of Escherichia coli is commercially available from Agilent (catalog reference number 200133). The ATCC 8739 strain is commercially available, and is particularly obtainable from ATCC. Its gene sequence is available in GenBank under the number GCA_016864475.1. Although not preferred, other E. coli strains known to those skilled in the art, particularly the K-12 strain (ATCC 10798), may be used instead of strain BL21 or ATCC 8739.

[0028] Advantageously, according to the present invention, the following calibration compositions are particularly preferred: - A calibration composition comprising a suspension of Escherichia coli strain BL21 in an aqueous liquid based on alcohol and / or acetonitrile. - A calibration composition comprising a suspension of Escherichia coli strain ATCC 8739 in an aqueous liquid based on alcohol and / or acetonitrile.

[0029] In the calibration composition according to the present invention, the alcohol and / or acetonitrile composition is selected for structural preservation of Escherichia coli present in the composition at a temperature of 0 to 37°C, particularly 0 to 25°C.

[0030] Particularly advantageously, this aqueous liquid based on alcohol and / or acetonitrile, or more generally the calibration composition, does not contain acids or other chemical substances that can react directly or indirectly with proteins. Preferably, chemical substances (surfactants, bases, etc.) that can extract proteins that are not usually detected in conventional deposits will be avoided.

[0031] In particular, in the calibration composition according to the invention, Escherichia coli is present at a concentration of 2 to 7 McF (McFarland), particularly 3 to 6 McF, for example 5 McF. Such concentrations can be determined by a densitometer (for example, DENSIMAT sold by bioMerieux) capable of measuring the optical density of the liquid composition under incident light at a wavelength of particularly 500 to 600 nm, preferably 550 nm.

[0032] The concentration of bacteria in the liquid composition can also be expressed in colony forming units per milliliter (cfu / mL). Thus, advantageously, in the calibration composition according to the invention, Escherichia coli is present at a concentration of 6×10 8 ~21×10 8 cfu / mL.

[0033] Table 1 below shows the correspondence between the optical density, the value in McFarland units (McF), and the bacterial concentration in 10 8 cfu / mL. TIFF2026520574000001.tif49170Table 1: Correspondence between McFarland units and cfu / mL

[0034] The calibration composition according to the invention is stable. This stability can be demonstrated by mass spectrometry, particularly MALDI-TOF. Thus, degradation of the proteins present, particularly the proteins selected as references for calibration, is not observed. Their masses do not change within the range of the analysis error. The stability of the calibration composition according to the invention can also be verified by its ability to provide a relatively constant and high-quality calibration over time.

[0035] Due to its good stability, the calibration composition of the present invention can be stored in a stoppered tube, particularly a plastic tube, or any other suitable airtight container. These can then be stored in this manner for several months, particularly at least one month, or at least two months, preferably at least six months, under temperature conditions of 0 to 37°C, typically 0 to 25°C. Advantageously, the composition of the present invention does not need to be stored in a frozen state at negative temperatures, particularly -18°C.

[0036] Therefore, without requiring special storage precautions, the calibration composition according to the present invention yields reliable and reproducible deposits on MALDI plates, thus enabling equally reliable and reproducible calibration, and thus mass spectrometry measurements, for the sample or microorganism to be characterized.

[0037] The calibration composition according to the present invention is a ready-to-use solution that can be easily used during mass spectrometry measurements and / or calibration procedures. Being a liquid formulation, it is easy to collect and deposit. The subsequent evaporation of the liquid phase results in localized deposits in the form of a thin, uniform cell layer. In particular, precise volumes of 1-2 μL, typically 1 μL, of the calibration composition according to the present invention can be easily and quickly collected and then precisely deposited on the deposit zone on the analysis plate. Because the calibration composition of the present invention is easily stored in a ready-to-use form, it simplifies the calibration workflow performed during mass spectrometry measurements by avoiding the need to perform fresh E. coli cultures daily.

[0038] According to another subject, the present invention relates to the use of a calibration composition according to the present invention as an external standard for calibrating a mass spectrometer according to the MALDI method, and more particularly according to the MALDI-TOF method.

[0039] The calibration composition according to the present invention can be used, for example, in internal calibration to adjust the acquisition parameters of a mass spectrometer according to the MALDI method, particularly the MALDI-TOF method, or for quality control.

[0040] In the context of the present invention, a method for calibrating a mass spectrometer suitable for the MALDI method, particularly the MALDI-TOF method, comprising the following steps: a) A step of providing a calibration composition according to the present invention, b) A step of depositing the calibration composition and a matrix suitable for the MALDI method onto at least one deposition zone of a MALDI analysis plate, and obtaining a control zone after drying, c) Placing the MALDI analysis plate in a mass spectrometer, obtaining a mass spectrum of the control zone over at least one determined mass-to-charge ratio range according to the MALDI method, particularly the MALDI-TOF method, and using the experimental values ​​obtained for the mass-to-charge ratio of the peaks in the spectrum corresponding to the ions of the so-called reference protein of the calibration composition as calibration values. d) A step of calculating the calibration relationship based on the theoretical mass-to-charge ratio and calibration value of the reference protein ions. Methods including this are also provided.

[0041] Advantageously, the determined mass-to-charge ratio range covers at least the range of 2000Th to 20000Th, preferably the range of 2000Th to 15000Th.

[0042] In a specific embodiment of the method according to the present invention, the theoretical mass-to-charge ratios used in step d) may be at least 6, preferably 8 to 50, and most specifically 8 to 32. According to one particular embodiment, when the strain of the calibration composition is the ATCC8729 strain of E. coli, the theoretical mass-to-charge ratios of the ions of the reference protein, and therefore of Th used, are selected from those listed in Table 2.

[0043] According to another specific embodiment, if the strain of the calibration composition is the BL21 E. coli strain, the ions of the reference protein, and therefore the theoretical mass-to-charge ratio at Th used, are selected from those listed in Table 5.

[0044] According to a specific embodiment, the theoretical mass-to-charge ratios used in step d) are at least 6, preferably 8 to 50, and are selected in particular from the following theoretical mass-to-charge ratios m / z at Th: - If the E. coli strain is BL21: 3128.18;3158.57;3179.59;3206.28;3579.84;3637.70;3936.51;4163.59;4185.35;4365.31;4438.61;4449.60;4497.60;4613.75;4768.45;4777.56;5069.74;5096.78;5150.50;5381.35;5461.10;5612.32;5725.60;6255.36 ;6298.04;6316.14;6411.55;6508.45;6547.83;6857.81;7158.68;7274.39;7707.56;7872.02;8326.18;8369.69;8876.22;8898.19;8994.20;9060.27;9226.49;9535.89;9554.12;9741.83;10138.48;10300.00;11223.63;11450.19 and 12227.19; - If the E. coli strain is ATCC 8739: 3128.18; 3158.57; 3206.28; 3579.84; 3637.70; 4163.59; 4185.35; 4365.31; 4438.61; 4613.75; 4768.45; 4777.56; 5069.74; 5096.78; 5150.50; 5381. 35;5612.32;6255.36;6316.14;6411.55;7158.68;7274.39;8369.69;8876.22;8994.20;9226.49;9535.89;9554.12;10138.48;10300.00;11223.63 and 11450.19.

[0045] More specifically, the theoretical mass-to-charge ratios used in step d) are at least 6, preferably 8 to 32, and particularly 32, and in particular, when the E. coli strain is BL21, they are selected from the following theoretical mass-to-charge ratios m / z at Th: 3128.18; 3158.57; 3206.28; 3579.84; 3637.70; 4163.59; 4185.35; 4365.31; 4438.61; 4613.75 ;4768.45;4777.56;5069.74;5096.78;5150.50;5381.35;5612.32;6255.36;6316.14;6411.55;7158.68;7274.39;8369.69;8876.22;8994.20;9226.49;9535.89;9554.12;10138.48;10300.00;11223.63 and 11450.19.

[0046] The MALDI matrix can be any matrix suitable for the MALDI method. In particular, such a matrix may contain one of the following compounds: 3,5-dimethoxy-4-hydroxycinnamic acid, α-cyano-4-hydroxycinnamic acid, ferulic acid, and 2,5-dihydroxybenzoic acid.

[0047] Finally, according to another aspect, the present invention relates to a method for characterizing a sample containing at least one microorganism based on the acquisition of a mass spectrum by the MALDI method, particularly the MALDI-TOF method, comprising the following steps: i) A step of depositing the sample and a matrix suitable for the MALDI method onto at least one deposition zone of an analysis plate, drying it, and obtaining a characterization zone. ii) A step of placing the analysis plate in a mass spectrometer suitable for the MALDI method, particularly the MALDI-TOF method, and generating a mass spectrum of the characterization zone. iii) A step of applying the calibration relationship calculated according to the calibration method of the present invention to the mass spectrometer and determining the mass-to-charge ratio of the peaks in the generated mass spectrum. Regarding how to implement it.

[0048] In particular, the control analysis zone and the characterization zone are part of the same MALDI analysis plate. This has the advantage of making the calibration operation simpler and faster by reducing handling steps.

[0049] In a specific embodiment, the characterization method according to the present invention includes identifying microorganisms present in a sample by comparing the mass-to-charge ratio values ​​of at least some peaks of the generated mass spectrum obtained in step iii) with reference values ​​characteristic of microorganisms.

[0050] Other objects, features, and advantages of the present invention will become apparent in light of the embodiments described below with reference to the following description and accompanying figures: [Brief explanation of the drawing]

[0051] [Figure 1] Figure 1 shows a schematic top view of a MALDI plate, specifically one sold by the applicant. [Figure 2] Figure 2 shows the time course of the MALDI-TOF mass spectrum obtained using the VITEK® MS system for an E. coli composition other than the present invention, in relation to Example 2, in which the bacteria are suspended whole in a commercially available solvent mixture (in this case, an HCCA matrix from bioMerieux; reference no. 411071). [Figure 3] Figure 3 shows the spectra obtained using a VITEK® MS system after storing two calibration compositions according to the present invention, each containing 70% v / v ethanol, at room temperature for 5 months, in relation to Example 3. These compositions were prepared according to two different protocols (A: derived from E. coli ATCC 8739 cultured on agar medium, B: derived from E. coli ATCC 8739 cultured in culture broth); [Figure 4]Figure 4 shows different MALDI-TOF spectra obtained using a VITEK® MS system, corresponding to the analysis of the contents of a tube initially containing 100 μL of the calibration composition according to the present invention (Escherichia coli ATCC 8739 suspended in an aqueous liquid containing 70% v / v ethanol) after evaporation of 20%, 50%, or 80% of the initial volume, in relation to Example 4; [Figure 5] Figure 5 shows the MALDI-TOF mass spectrum obtained using a VITEK® MS system before calibration for the calibration composition of Example 3 (Escherichia coli BL21 suspended in an aqueous liquid containing 70% v / v ethanol), in relation to Example 6; [Figure 6] Figure 6 shows two graphical representations of the errors attributable to the peak (m / z) selected as the reference for calibration, obtained for the calibration composition according to the present invention (Escherichia coli BL21 suspended in an aqueous liquid containing 70% v / v ethanol), in relation to Example 7, for a newly prepared composition (T0) or for a composition stored at 2-8°C for 9 or 12 months (T9, T12). The relevant MS spectra were obtained using a VITEK® MS system before calibration (A) and after calibration (B); [Figure 7] Figure 7 shows the first supercalibration graph used in Example 8; [Figure 8] Figure 8 shows the second final supercalibration graph from 49 theoretical masses selected from 6563 MALDI-TOF spectra obtained using the VITEK® MS system used in Example 8. [Examples]

[0052] Example 1: Preparation of the calibration composition according to the present invention, deposition on a MALDI analysis plate, and calibration The calibration composition according to the present invention can be prepared by simply placing a selected amount of a selected strain of total Escherichia coli in an alcohol and / or acetonitrile-based liquid. Thus, the calibration composition comprises a suspension of stored total Escherichia coli. Depending on the amount of calibration composition to be prepared, the Escherichia coli used may be directly purchased or derived from bacteria purchased and cultured according to any suitable technique, such as on a petri dish or in broth. These culture techniques are well known to those skilled in the art. As demonstrated in the examples, the culture method does not affect the stability of the resulting calibration composition.

[0053] As an example of a method for preparing a calibration composition according to the present invention, Escherichia coli is cultured in broth in a fermenter until a desired amount of biomass is achieved. The biomass is then pelletized by centrifugation and then diluted with a 70% ethanol solution to obtain the desired concentration. Mechanical dispersion operations may prove useful to promote homogenization of the solution and to accurately quantify the bacterial concentration. This can be achieved, for example, by creating a vortex or dispersing the bacteria using an apparatus such as the Ultra-Turrax from IKA (Stauffen, Germany) equipped with a high-performance rotor-stator. A sonication process can also be performed. In any case, care must be taken to minimize the degradation of bacterial cells to avoid membrane wall rupture and the formation of cell debris. Despite precautions taken, some bacteria may degrade, and cell debris may be found in the calibration composition according to the present invention. Once the solution is homogenized and adjusted to the desired concentration, e.g., 5 McF, it is simply a matter of distributing it into appropriate containers for easy use.

[0054] The calibration composition according to the present invention may be used directly, stored in any suitable container, and / or sold as a composition ready for immediate use after being stored in any suitable container. Suitable containers include, in particular, plastic stoppered tubes or any type of suitable sealed container. The container has a capacity selected to contain a desired amount of calibration composition, specifically 10 to 300 μL, typically 200 μL. Such a capacity allows for the generation of numerous deposits, usually 1 μL deposits, and thus enables numerous calibrations.

[0055] Preliminary stability studies presented in the examples have shown that the calibration composition according to the present invention is stable for at least one year at 2-8°C, at least two months at room temperature (typically 18-25°C), and at least one day at room temperature when the container is open. Therefore, the calibration composition according to the present invention is stable over time, enabling the acquisition of good calibration.

[0056] During use, the calibration composition according to the present invention is deposited in the control zone of the MALDI plate. This deposition can be performed using pipetting techniques or, in some cases, spraying techniques. A drop of the calibration composition, for example, about 1-2 μL, can be deposited to cover the entire deposition zone. Because it is a liquid formulation, it is easy to form a thin, uniform layer of the calibration composition on the plate.

[0057] No special expertise or skills are required to use the calibration compositions according to the present invention. They are compatible with the manual and automated preparation of MALDI plates, particularly VITEK® MS-DS type MALDI plates (for example, using Colibri® technology provided by the applicant).

[0058] The operation of depositing the calibration composition and matrix suitable for the MALDI method according to the present invention onto a MALDI plate can be performed sequentially, or by depositing a mixture of the calibration composition and matrix.

[0059] Advantageously, the procedure would be carried out as follows: deposition of the calibration composition, deposition of the MALDI matrix, and then drying. The calibration composition according to the present invention can be easily deposited by pipetting 1 μL onto the control zone of a MALDI plate, followed by the addition of 1 μL of MALDI matrix, particularly HCCA matrix, and drying. The matrix used in the MALDI method is generally an organic molecule having specific properties. They must be able to absorb the energy of the laser used for irradiation, resulting in the vaporization of the matrix and the formation of ions. Furthermore, the matrix must be compatible with the sample being analyzed and not interfere with the ions of interest. Generally, the matrix used in the MALDI method is photosensitive and crystallizes in the presence of a population of microorganisms while maintaining the integrity of the molecules present. Such matrices, particularly those suitable for MALDI-TOF mass spectrometry techniques, are well known. These consist of compounds selected from, for example, 3,5-dimethoxy-4-hydroxycinnamic acid, α-cyano-4-hydroxycinnamic acid (present in HCCA matrix), ferulic acid, and 2,5-dihydroxybenzoic acid. Many other compounds are known to those skilled in the art. Liquid matrices also exist that do not crystallize at atmospheric pressure or under reduced pressure. Any other compounds can be used that enable the ionization of molecules present in the characterization zone under the influence of a laser beam. Each matrix has specific properties suitable for different types of samples and analyses.

[0060] For the formation of the matrix, such compounds are typically dissolved in water, preferably "ultrapure" grade water, or a water / organic solvent mixture. Examples of conventionally used organic solvents include acetone, acetonitrile, methanol, or ethanol. The matrix may contain trifluoroacetic acid to facilitate ionization. The matrix is ​​deposited directly onto the control zone, covering the calibration composition or mixing it with the calibration composition.

[0061] Drying of the deposited calibration composition on the MALDI plate, either before or after matrix deposition, is carried out at a temperature and time suitable to ensure the formation of a thin, uniform layer. Mild conditions well known to those skilled in the art are preferred. Drying of the calibration composition can be carried out by evaporation, either alone or with the matrix added, by exposing the whole to ambient air for several minutes. This evaporation also ensures the crystallization of the present matrix.

[0062] Advantageously, the amount of E. coli in the calibration composition deposited per control zone is generally at least 10 5 cfu, especially 6×10 5 ~42×10 5 It is a CFU.

[0063] Once the MALDI plate is prepared with a layer of calibration composition in the form of a control zone as described above, it is placed in a mass spectrometer, particularly a MALDI or MALDI-TOF mass spectrometer, and the corresponding mass spectrum is acquired. This involves irradiating the MALDI plate with a MALDI laser, resulting in the desorption and ionization of molecules present at the irradiated spots. The desorbed and ionized proteins are then detected to generate a mass spectrum. Thus, the acquisition of the mass spectrum is performed by irradiating the MALDI plate with a MALDI laser and detecting the desorbed and ionized proteins.

[0064] In particular, any type of MALDI-TOF mass spectrometer can be used to generate the mass spectrum. Such an instrument is configured as follows: i) An ionization source (usually a UV laser) intended to ionize the control zone, thereby ionizing the calibration composition and / or the sample present in the characterization zone; ii) Accelerators for ionizing molecules by applying a potential difference; iii) A tube under reduced pressure through which ionized and accelerated molecules move; iv) A mass spectrometer designed to separate formed molecular ions as a function of the mass-to-charge ratio (m / z); v) A detector designed to measure signals directly generated by molecular ions.

[0065] The laser beam used for ionization can have any wavelength that is favorable for the sublimation or vaporization of the matrix. Preferably, ultraviolet or infrared wavelengths will be used. This ionization can be carried out using, for example, a nitrogen laser emitting an ultraviolet beam at 337 nm (e.g., used in VITEK® MS systems) or a neodymium-doped yttrium lithium fluoride (Nd:YLF) laser emitting at 349 nm (e.g., used in VITEK® MS PRIME systems).

[0066] The time of flight for an ion to reach the detector is used to calculate the ion's mass. In this way, a mass spectrum is obtained in which the intensity of the signal corresponding to the number of ionized molecules with a given mass-to-charge ratio [m / z] is expressed as a function of the m / z ratio of the molecules colliding with the detector. The mass-to-charge ratio [m / z] is expressed as Thomson (Th).

[0067] In particular, the mass spectrum is generated by detecting at least some of the molecules that are ionized and accelerated, and then can move freely within a tube under reduced pressure. The time it takes for the molecules to travel through the tube under reduced pressure is measured, and a signal corresponding to the number of ionized molecules that reach the spectrometer's detector at a given moment is obtained. Thus, an uncalibrated spectrum is obtained with the time of flight of the ions on the x-axis and the intensity of the observed signal on the y-axis. This is converted into a mass spectrum by calculating the mass-to-charge ratio (m / z) corresponding to the time of flight of the detected molecules. To do this, a time-of-flight calibration formula is established using a reference molecule. As a result, a calibrated mass spectrum is obtained, as a function of the m / z ratio of the detected molecules, corresponding to the signal of the number of ionized molecules with a given mass-to-charge m / z.

[0068] During the calibration process, peaks corresponding to the so-called reference molecules (called reference peaks) in the ionized form of the calibration composition (called ions of the reference molecule) are identified, and their theoretical mass-to-charge ratios (m / z) are used as calibration values. This identification can be performed by comparing the measured flight time (called experimental flight time) of the peaks in the uncalibrated spectrum with the theoretical mass of the ions of the reference protein, or, if a previous calibration is already available and applied in the instrument, by comparing it with the measured mass-to-charge ratio (m / z) of the mass spectrum (called experimental mass-to-charge ratio (m / z)). Once these calibration values ​​are identified, the calibration relationships and errors are calculated. Various linear or nonlinear regression methods can be used to calculate such calibration relationships. Examples include the least squares method or any method well known to those skilled in the art for performing calibration. Generally, mass spectrometers are equipped with appropriate software for implementing such methods. Considering the experimentally obtained values ​​and the corresponding theoretical values, an error in parts per million (ppm) is calculated for each reference mass-to-charge ratio value (or reference protein ion). To do this, the following formula can be used: (Theoretical mass - Experimental mass) / Theoretical mass × 10 6 For the selected set of reference protein ions, the mean error, which corresponds to the mean value of the errors expressed as absolute values, can be calculated. The smaller the mean error, the better the calibration.

[0069] Next, the calibration relationships thus established are applied to determine the mass-to-charge ratio of the ion peaks of proteins or molecules present in the analyte on the characterization zone of the MALDI plate. These corrected masses (also called calibration masses) are obtained in step iii) of the method for characterizing the sample, as defined above.

[0070] In summary, the calibration composition and method for calibrating a mass spectrometer according to the present invention enable accurate measurement of the mass of the ions being analyzed, and thus enable reliable identification of microorganisms. The calibration composition and method according to the present invention are easy to implement and can be used for a variety of analytical applications, particularly for the identification of microorganisms by mass spectrometry using the MALDI method, and especially the MALDI-TOF method. In the context of the present invention, the MALDI-TOF analysis may be a simple MALDI-TOF analysis or a MALDI-TOF-TOF analysis.

[0071] The methods according to the present invention (calibration methods and methods for characterizing samples) are computerized or automated methods. In the methods according to the present invention, steps such as acquisition, calculation, comparison, and determination are performed by a computer or electronic device programmed to perform the steps, which is incorporated into the spectrometer used. Specific software can be developed and installed on the computer or electronic device used to perform the calibration method, and in particular to obtain calibration relationships. In particular, this software is programmed to handle necessary functions, such as acquiring mass spectrometry data and calculating the mass-to-charge ratio of mass peaks in the mass spectrum corresponding to ions of unknown molecules or proteins.

[0072] To identify peaks corresponding to calibration values ​​on the mass spectrum of a calibration composition, calibration software can be programmed to analyze mass spectrometry data and identify peaks corresponding to calibration values. This process may include adaptive peak detection, mass-to-charge ratio comparison, and thresholding algorithms to accurately identify the relevant peaks.

[0073] In the calibration relationship calculation, once the peak corresponding to the calibration value is identified, the calibration software performs calculations to determine the calibration relationship. This calculation can use different statistical methods, such as linear regression or nonlinear regression, to establish a formula derived from the theoretical mass-to-charge ratio of the ion of the reference protein and the measured mass-to-charge ratio for the peak corresponding to the ionized reference protein used as the calibration value.

[0074] Regarding the application of calibration relationships, once determined, the software applies them to convert the measured (also called experimentally) time-of-flight or mass-to-charge ratios of peaks corresponding to unknown entities on the mass spectrum obtained for the sample to be analyzed into calibrated mass-to-charge ratios, which are then plotted on the generated mass spectrum. This process makes it possible to obtain accurate measurements of the mass-to-charge ratios of ions present in the sample being analyzed.

[0075] The samples to be analyzed may have various origins. Examples include biological origins, particularly animal or human samples. Such samples may correspond to biological fluid samples such as whole blood, serum, plasma, urine, cerebrospinal fluid, organic secretions, or tissue swabs or isolated cells. These samples may be deposited as is, or preferably, before being deposited on MALDI plates, they may be subjected to concentration or preparation of culture types, concentration and / or extraction or purification steps according to methods known to those skilled in the art. Samples may also be agricultural products such as meat, milk, and yogurt, other potentially contaminated consumer products, or cosmetics or pharmaceuticals. Here again, such products may be subjected to concentration or preparation of culture types, concentration and / or extraction or purification steps before being deposited on MALDI plates. Samples to be analyzed are typically obtained from cultures in broth or on agar plates to concentrate the microorganisms to be sought. For example, a drop of biomass or microbial suspension can be deposited directly into ultrapure water or buffer.

[0076] The following embodiments illustrate the present invention with reference to several accompanying figures, but are not intended to limit it.

[0077] In the following examples, optical density is measured using a DENSIMAT densitometer (bioMerieux, France) in accordance with the recommendations in the user manual (reference 99535, version C, published in 2007).

[0078] Example 2: Evaluation of the stability of a suspension of E. coli in a commercially available solvent mixture (bioMerieux; reference number 411071). Commercially available E. coli strain ATCC 8739, purchased from ATCC, was added to an α-cyano-4-hydroxycinnamic acid matrix (referred to as the HCCA matrix), consisting of 333 ml of ethanol, 333 ml of acetonitrile, 333 ml of water, 30 ml of trifluoroacetic acid, and 31 g of HCCA matrix (bioMerieux; reference no. 411071), until a concentration of 5 McF (corresponding to an optical density of 1.0-1.1 at 550 nm) was obtained. The HCCA matrix is ​​commonly used in MALDI-TOF mass spectrometers for the measurement of peptides and proteins.

[0079] Next, the stability of the suspension was analyzed using a MALDI-TOF mass spectrometer (VITEK® MS, bioMerieux) according to the change in the calibration peak between T0 and T7 days (T0 to 7 days later). A gradual change in the peak shape was observed across all spectra.

[0080] For example, the characteristic double-charged peak of the DNA-binding protein HUα was clearly visible at T0 (m / z 4767). However, a third peak appeared at T24h (1 day after T0), a fourth peak at T48h (2 days after T0), and eight peaks were detected at T7d (7 days after T0) (Figure 2).

[0081] Thus, given that degradation was observed over time, and considering the mixture of solvents used (ethanol, acetonitrile, and trifluoroacetic acid), the degradation appears to be of chemical origin rather than biological origin. Considering that the observed delta between different peaks in the mass-to-charge ratio of the ionized protein over time was approximately 28 Da, and that the presence of ethanol and trifluoroacetic acid can protonate the protein, the inventors believe that the degradation likely corresponds to ethylation.

[0082] As a result, the solvent mixture in the HCCA matrix commonly used in MALDI-TOF instruments cannot be used as a solvent for calibration solutions because it cannot achieve sufficient stability over time for the calibration peak of the E. coli strain.

[0083] Example 3: The method of culturing the strain does not affect the performance of the calibration composition according to the present invention. The two calibration compositions according to the present invention were prepared from the ATCC 8739 Escherichia coli strain used in Example 2, but were cultured according to two different methods.

[0084] A. Protocol for preparing a calibration composition from the ATCC 8739 strain obtained by growth on solid agar medium in a Petri dish: - Incubate the strain in a Petri dish on solid agar medium (bioMerieux; reference no. 43039) at 37°C for 18-24 hours. - Dilute the strain to 5 McF with 70% (v / v) ethanol (i.e., 70 vol% ethanol and 30 vol% water) (i.e., obtain an optical density of 1.0-1.1 at 550 nm) to obtain a calibration composition. - The composition is filled into tubes (50 μL, 100 μL, and 200 μL). - Store the tubes at room temperature (18-24°C) and 2-8°C.

[0085] When using a calibration composition, 1 μL of the composition and 1 μL of the HCCA matrix are deposited on a MALDI plate.

[0086] Protocol for preparing a calibration composition from the same ATCC 8739 Escherichia coli strain obtained by B. broth culture: - Incubate in Triptycase soybean culture broth (bioMerieux; reference number 42100) at 37°C for 18-24 hours. - Centrifuge at 4500 rpm for 10 minutes to concentrate the cells into a pellet. - Rinse the pellets twice with water. - The pellet is resuspended in a 70% ethanol solution and diluted to obtain a calibration composition according to the present invention containing E. coli concentrated to 5 McF (corresponding to an optical density of 1.0 to 1.1 at 550 nm) in 70% ethanol. - The composition is filled into tubes (50 μL, 100 μL, and 200 μL). - Store the tubes at room temperature (18-24°C) or 2-8°C.

[0087] When using a calibration composition, 1 μL of the composition and 1 μL of the HCCA matrix are deposited on a MALDI plate.

[0088] Subsequently, the calibration compositions prepared according to the two protocols described above were used to calibrate a MALDI-TOF mass spectrometer (VITEK® MS, bioMerieux). Both compositions consistently showed reference peaks (including the calibration values ​​used during calibration) and did not degrade (data not presented), thus demonstrating that very good calibration of the mass spectrometer was possible and that the method of producing the strain (cultivation on a petri dish or in culture broth) does not affect the quality of the calibration.

[0089] Furthermore, no degradation was observed in the peaks of various spectra even after storage at room temperature for 5 months (Figure 3). In other words, the peaks observed in Figure 3 represent the expected mass for strain ATCC 8739, as shown in Table 2. In contrast to the formulation in Example 2, no degradation was observed, and the peak masses remained constant within the range of analytical error. This analytical error is inherent to the MALDI-TOF method. The results obtained with storage at 2–8°C were similar.

[0090] As a result, the method used to culture the E. coli strains to obtain the calibration composition does not affect the calibration quality in the MALDI-TOF mass spectrometer, nor the temporal stability of the composition.

[0091] Example 4: Evaluation of the effect of evaporation on the quality of the calibration composition according to the present invention Calibration composition used: Escherichia coli ATCC strain 8739 was suspended in a 70% ethanol solution at a concentration of 5 McF (i.e., obtaining an optical density of 1.0-1.1 at 550 nm) and stored in 50 μL, 100 μL, 200 μL, and 300 μL tubes.

[0092] The tubes were left open at room temperature (24°C ± 1°C) until 20%, 50%, or 80% of the initial volume evaporated. Various partial evaporation calibration compositions were then used as calibration solutions for MALDI-TOF mass spectrometers (VITEK® MS, bioMerieux).

[0093] After analysis, all tested compositions, regardless of initial volume and degree of solvent evaporation, allowed for very good calibration of the mass spectrometer. Various MALDI-TOF spectra for 100 μL tubes with evaporation rates of 20%, 50%, or 80% are shown in Figure 4. Aside from the analytical errors inherent in the MALDI-TOF method, the spectra are very similar: the peaks listed in Table 2 are observed at their expected masses; therefore, these allow for spectral calibration.

[0094] Example 5: Preparation of a calibration composition according to the present invention for Escherichia coli BL21 in 70% ethanol Similar to Example 3, the calibration composition according to the present invention was prepared using preparation protocol A with E. coli strain BL21 (E. coli BL21 suspended in 70% v / v ethanol).

[0095] As demonstrated in Example 6 below, this solution enabled excellent calibration results in a MALDI-TOF mass spectrometer.

[0096] Example 6: Calibration of a MALDI-TOF apparatus using the calibration composition according to the present invention Generally, MALDI-TOF mass spectrometers measure the time of flight of ions in a vacuum after they have been accelerated by an electric field. The time of flight is determined by the law of kinetic energy: TIFF2026520574000002.tif5170[where, E c [where m is kinetic energy, m is mass, and V is the velocity of the ion], it is related to the mass of the ion.

[0097] Therefore, the mass of an ion can be determined by measuring its time of flight and establishing a relationship between these two physical quantities, generally using a quadratic equation, or quadratic polynomial.

[0098] In some cases, higher-order polynomials may be used to account for the physical and electronic shortcomings of the mass spectrometer. In practice, the equations are calibrated using molecules of known mass to account for experimental variations inherent in the instrument, the MALDI plate on which the sample is deposited, or the acquisition conditions (temperature, pressure in the vacuum tube, etc.).

[0099] This device may be delivered by the manufacturer with factory calibrations pre-stored. This allows the user to directly use Thomson (Th) values ​​to acquire and analyze the initial spectrum. Thomson is a unit corresponding to the value obtained by dividing the mass of an ion by its charge. Subsequently, the device can be periodically recalibrated using the Th spectrum without relying on time-of-flight values. Therefore, the user becomes accustomed to adjusting the device and visualizing the spectrum using mass rather than time-of-flight.

[0100] The calibration quality can be easily evaluated by calculating the average error in the mass-to-charge ratio of the reference protein ions. This average error is calculated using the absolute value of the error.

[0101] For example, MALDI-TOF mass spectra obtained using a VITEK® MS spectrometer from bioMerieux were calibrated using a calibration composition prepared according to Example 5.

[0102] The instrument's acquisition range was set to between 2000 and 20000 Th, which is the typical mass-to-charge ratio range for identifying microorganisms in a microbiology laboratory. The resulting spectra of the calibration composition show uncalibrated experimental m / z values ​​and are shown in Figure 5.

[0103] The spectra were then calibrated using a set of mass-to-charge ratios of protein ions present in E. coli, extracted from Table 2. In the "Ion Charge" column of Table 2, 's' indicates an ion with one charge, and 'd' indicates an ion with two charges. Proteins show post-translational modifications for proteins with given names and sequences. In the "Post-translational Modification" column, 'c' corresponds to the cleavage of the N-terminal methionine, and 'm' corresponds to the methylation of the second amino acid (which becomes the terminal) after the cleavage of the N-terminal methionine. The ion names of the reference protein are given to combine the protein name, its charge, and post-translational modifications corresponding to the NCBI sequence. TIFF2026520574000003.tif131170 Table 2: 32 selected theoretical m / z values ​​for E. coli ATCC 8739

[0104] To calibrate the spectrum using the least squares method with a quadratic equation, the ions of the reference protein are searched for within an acceptable range of 800 ppm.

[0105] The average error before calibration was 372.69 ppm. This average decreased significantly to 68.50 ppm after the first calibration, thus demonstrating that the mass-to-charge ratio recalibrated with the composition according to the present invention (the "After First Calibration" column in Table 3) is more accurate than the pre-calibration value.

[0106] To further improve the calibration, a second calibration can be performed after reducing the tolerance to 300 ppm. This operation eliminates some erroneous associations that could slightly distort the first calibration. These erroneous associations correspond to peaks that were incorrectly attributed to specific theoretical masses when considering a large error tolerance (800 ppm). This second calibration slightly improves the measurement accuracy by considering only peaks with errors less than 300 ppm. The average error is 63.18 ppm.

[0107] Interestingly, it is not necessary to find all mass-to-charge ratios explored in the mass spectrum. For example, peak 28 is not observed in the spectrum before recalibration. After the first recalibration, peaks 23 and 27 are beyond the acceptable range and are therefore not used in the second calibration. Thus, not all peaks need to be present to ensure a good calibration. This method is robust to the absence of one or more peaks. This property is particularly important for calibrating MALDI-TOF spectra, as the MALDI-TOF method has a stochastic behavior that can randomly cause the disappearance or mass shift of certain peaks. For a given sample, certain peaks may be visible or not visible with each acquisition, depending on the crystal sublimated by the laser beam. Also, some peaks may have more or less noisy shapes, which can introduce more or less significant errors into the detected mass. TIFF2026520574000004.tif140170 Table 3: Pre- and post-calibration m / z readings of the calibration composition (Escherichia coli BL21; 70% v / v ethanol) according to the present invention, using 32 theoretical m / z values ​​for E. coli ATCC 8739 shown in Table 2.

[0108] Example 7: Stability test of the calibration composition according to Example 5 The possibility of calibration using a time-stable calibration composition according to Example 5 was established by verifying that it is possible to calibrate a VITEK® MS MALDI-TOF mass spectrometer using a batch of this calibration composition newly prepared according to Example 5 (condition T0), and then calibrate the VITEK® MS MALDI-TOF mass spectrometer using the same batch of calibration composition stored at 2-8°C for 9 months (condition T9) or 12 months (condition T12).

[0109] To simulate intensive usage conditions, batches stored at 2–8°C for 9 or 12 months were left open for 1 hour and 30 minutes to allow some of the solvent to evaporate, and then closed again and left at room temperature (18–25°C) for 180 hours.

[0110] The mass-to-charge ratios before and after calibration, obtained using a list of 32 reference peaks, are shown in Tables 4, 4bis, and 4ter below. The measurement errors of the calibration peaks are plotted in Figure 6 (A: before calibration, B: after calibration).

[0111] Excellent calibration was achieved, characterized by a significant decrease in the mean error at T0, T9, and T12, from 157.69 ppm to 17.64 ppm, from 186.45 ppm to 19.45 ppm, and from 287.29 ppm to 29.01 ppm, respectively.

[0112] These results demonstrate excellent calibration with liquid solutions from T0 to 12 months, despite simulations of intensive use with an evaporation time of 1 hour 30 minutes and a duration of 7.5 days at room temperature. Interestingly, the improvement in calibration quality is achieved even without the observation of the presence of several peaks / values ​​(denoted as NA). Table 4: Pre- and post-calibration m / z readings of a newly prepared calibration composition according to the present invention (Escherichia coli BL21; 70% v / v ethanol), referencing 32 theoretical m / z values ​​for E. coli ATCC 8739. Table 4bis of TIFF2026520574000006.tif140170: m / z readings before and after calibration of the calibration composition according to the present invention (Escherichia coli BL21; 70% v / v ethanol) stored at 2-8°C for 9 months. Table 4ter of TIFF2026520574000007.tif140170: m / z readings before and after calibration of the calibration composition according to the present invention (Escherichia coli BL21; 70% v / v ethanol) stored at 2-8°C for 12 months.

[0113] Example 8: Selection of calibration values ​​according to the strain used As demonstrated in Example 6, a calibration suspension prepared using E. coli strain BL21 allows for improved calibration during MALDI-TOF spectrum acquisition. However, the quality of the calibration depends on the reference protein selected for calibration.

[0114] The objective of this example is to present a methodology for selecting a reference protein with a known theoretical mass-to-charge ratio that will be used for calibration in a given strain (in this case, E. coli strain BL21). This methodology can be applied to all other strains of E. coli.

[0115] Generally, interpreting MALDI-TOF spectra involves searching for a list of expected theoretical mass-charge ratios from experimental values, i.e., mass-charge ratios measured from uncalibrated mass spectra. This association occurs when the value falls below a certain precision threshold, i.e., an acceptable range or error expressed in "ppm," meaning "parts per million."

[0116] A) Selection of the theoretical mass-to-charge ratio of E. coli strain BL21 The selection of theoretical mass-to-charge ratios is performed over the m / z range of 3000 to 12500 Th (which is trivially fixed with respect to the capabilities of the mass spectrometer), with the aim of selecting proteins and associated ions such that the corresponding theoretical mass-to-charge ratios are uniformly distributed within this range.

[0117] Table 5 below shows the theoretical mass-to-charge ratio (m / z) and corresponding name for several pre-selected proteins, following the same rules as Table 2. Although not listed in Table 2, post-translational modifications may also be of the type of formylation or acetylation of the second amino acid after cleavage of the N-terminal methionine; these modifications are denoted as f or a. TIFF2026520574000008.tif105170 Table 5: 49 theoretical m / z values ​​of E. coli BL21

[0118] (B) Experimental MALDI-TOF spectrum In its database, the applicant possesses 14,332 available experimental MALDI-TOF spectra of E. coli BL21 obtained over a long period (6,614 obtained using a VITEK® MS mass spectrometer and 7,718 obtained using a VITEK® MS PRIME mass spectrometer).

[0119] Of these spectra, 52 are excluded because they allow less than half of the theoretical mass-to-charge ratio being searched, thus ultimately retaining 14,280 experimental MALDI-TOF spectra.

[0120] C) Verification of the theoretical mass-to-charge ratio used for calibration C1. First verification of the theoretical mass-to-charge ratio Before using the interpretation results, the quality of the calibration was verified. This is because insufficient calibration can distort the interpretation of experimental peaks, particularly due to irregular precision.

[0121] For example, a "shift" in the theoretical mass-to-charge ratio can systematically bias the calibration, or an "unstable" theoretical mass-to-charge ratio can disrupt the overall accuracy of the calibration. In reality, these two situations conflict, so it is necessary to exclude theoretical masses that could negatively impact the calibration.

[0122] For this reason, only a portion of the spectrum was used. The association of 7,724 MALDI-TOF spectra with 49 selected theoretical masses resulted in 295,829 interpretations, or approximately 38 interpretations per spectrum, meaning that 38 of the 49 explored observed theoretical or "experimental" mass-to-charge ratios were obtained.

[0123] To grasp the overall picture of the calibration, the overall interpretation of each theoretical mass-to-charge ratio was displayed on the same graph called the supercalibration graph. As shown in Figure 7, the median of the interpretation error for each theoretical mass-to-charge ratio was displayed along with the variance interval corresponding to the standard deviation of the error.

[0124] From the supercalibration graph in Figure 7, it became possible to exclude the theoretical mass-to-charge ratio that negatively affects calibration using three criteria: - Visibility of theoretical mass-charges: Some theoretical mass-charges are observed very rarely and are therefore excluded from calibration. This applies to the four theoretical mass-charge ratios. - Shift in mass-to-charge ratio: Since some theoretical mass-to-charge ratios appear to be abnormally shifted, a threshold of 100 ppm is applied to the median of the error. This criterion is applied to seven theoretical mass-to-charge ratios. - Mass stability: Some theoretical mass-to-charge ratios allow for higher standard deviations and variance intervals. Therefore, a threshold of 100 ppm is applied to the standard deviation so that theoretical mass-to-charge ratios with standard deviations greater than 100 ppm can be excluded. This criterion is applied to six theoretical mass-to-charge ratios.

[0125] By combining the three criteria, twelve different theoretical mass-to-charge ratios were excluded for calibration, and are shown in Table 6 below. TIFF2026520574000009.tif55170 Table 6: Twelve m / z values ​​of calibration compositions (E. coli BL21, 70% v / v ethanol) according to the present invention that were excluded from the calibration process.

[0126] At the end of the first verification, 37 theoretical mass-to-charge ratios were retained.

[0127] C2. Second verification of the theoretical mass-to-charge ratio Next, for each theoretical mass-to-charge ratio, we analyzed 7650 spectral samples to obtain a supercalibration graph showing the median interpretation error and the variance interval corresponding to the standard deviation of the error.

[0128] Next, a threshold of 40 ppm was applied to the median error. This threshold allowed for the exclusion of five theoretical mass-to-charge ratios for calibration, which are listed in Table 7 below. TIFF2026520574000010.tif28170 Table 7: Five m / z values ​​of calibration compositions (Escherichia coli BL21, 70% v / v ethanol) according to the present invention that were excluded from the calibration process.

[0129] At the end of the second verification, 32 theoretical masses were retained for calibration, while the remaining 17 were explored to improve control. This will be discussed in the next section.

[0130] D) Final interpretation method 6563 spectra acquired with a VITEK® MS spectrometer were studied.

[0131] Prior to interpretation, the spectra were calibrated using 32 pre-selected theoretical masses. The calibration consisted of two consecutive quadratic regressions using the 32 selected calibrated theoretical mass-to-charge ratios, with tolerance thresholds of 500 ppm and 300 ppm, respectively.

[0132] Finally, a total of 49 (theoretically calibrated and added) mass-to-charge ratios were explored with an acceptable threshold of 300 ppm.

[0133] E) Results As mentioned above, to understand the calibration from a general perspective, the overall interpretation of each theoretical mass-to-charge ratio is displayed on the same graph. As shown in Figure 8, the median interpretation error for each theoretical mass-to-charge ratio is displayed along with the confidence interval corresponding to the standard deviation of the error.

[0134] Next, the interpretation performance was calculated for the 32 mass-to-charge ratios used as calibration values ​​and for the additional set of 17 mass-to-charge ratios. The results are shown in Table 8 below. TIFF2026520574000011.tif36170 Table 8: Calibration performance according to the present invention

[0135] As is clear from Table 8, firstly, the 32 mass-to-charge ratios used in correspondence with the calibration values ​​were very frequently observed, with an average visibility rate of 95% across all spectra.

[0136] Secondly, the absolute measurement error was also corrected by calibration, with the median decreasing from 119.48 ppm to 19.41 ppm, the mean decreasing from 152.48 ppm to 25.23 ppm, and the standard deviation decreasing from 126.39 ppm to 23.50 ppm. In other words, the method of verifying the mass-to-charge ratio corresponding to the calibration value made it possible to provide an interpretation that is six times more accurate and four times less uncertain in measurement. Thus, the method of selecting the reference theoretical mass-to-charge ratio made it possible to significantly improve calibration performance.

[0137] Furthermore, a mass spectrometer calibrated in this way can be successfully used to identify different microorganisms.

[0138] Example 9: Stability and performance of the calibration composition according to the present invention Table 9 below provides, as an indicator, the results of time-dependent stability tests conducted on various calibration compositions according to the present invention.

[0139] The compositions tested were prepared by suspending E. coli BL21 bacteria in a pure alcohol solution, or in an aqueous alcohol or acetonitrile solution: - 100% (v / v) ethanol solution, - 20% (v / v) ethanol solution, - 50% (v / v) ethanol solution, - 70% (v / v) ethanol solution, - 70% (v / v) isopropanol solution, and - 70% (v / v) acetonitrile solution.

[0140] The stability of these calibration compositions was evaluated according to the obtained calibration performance. This is expressed as the average of the errors associated with the 32 calibration masses selected in Example 8.

[0141] Measurements were performed using either a newly prepared (T0) calibration composition or a calibration composition stored at room temperature for 8 days (T8). TIFF2026520574000012.tif32170 Table 9: Stability of calibration compositions in terms of calibration performance (expressed as the average of errors).

[0142] Therefore, the calibration performance of the six calibration compositions reported in Table 9 allows for a reduction in the average error by approximately six times at T0 and approximately ten times at T8, which demonstrates the superior calibration according to the present invention.

[0143] For comparison, calibration performed using freshly cultured E. coli ATCC 8739 bacteria, as specified in the VITEK® MS user manual, yielded an average error of the order of 30 ppm after calibration (according to recent measurements performed under the same conditions as in Example 8, the pre-calibration reading was 109.94 ppm and the post-calibration reading was 26.9 ppm).

[0144] It should also be noted that the mass spectrum obtained from the Escherichia coli BL21 bacteria suspended in a 70% formic acid solution had too few peaks to be calibrated.

Claims

1. Calibration composition for mass spectrometers, comprising all E. coli suspended in an alcohol and / or acetonitrile-based liquid [having sterilizing properties, being volatile, and non-denaturing / non-reactive to bacterial proteins].

2. The calibration composition according to claim 1, characterized in that the liquid is alcohol-based.

3. The calibration composition according to claim 2, characterized in that the liquid is an aqueous liquid having an alcohol content of at least 20% v / v.

4. The calibration composition according to claim 2 or 3, wherein the alcohol is ethanol and / or isopropanol.

5. The calibration composition according to claim 3 or 4, wherein the liquid is a water / alcohol mixture having an alcohol content of 70% v / v, preferably a water / ethanol mixture.

6. The calibration composition according to claim 1, wherein the liquid is an aqueous mixture of water / acetonitrile.

7. The calibration composition according to any one of claims 1 to 6, wherein the Escherichia coli is a single strain.

8. The calibration composition according to any one of claims 1 to 7, wherein the Escherichia coli is strain BL21 or strain ATCC 8739.

9. E. coli in the calibration composition 6 × 10 ８ ~21 x 10 ８ A calibration composition according to any one of claims 1 to 8, present at a concentration of cfu / mL.

10. A method for calibrating a mass spectrometer suitable for the MALDI method, the following: a) A step of providing a calibration composition according to any one of claims 1 to 9, b) A step of depositing the calibration composition and a matrix suitable for the MALDI method onto at least one deposition zone of a MALDI analysis plate, and obtaining a control zone after drying. c) Placing the MALDI analysis plate in a mass spectrometer, obtaining a mass spectrum of a control zone over at least one determined mass-to-charge ratio range according to the MALDI method, particularly the MALDI-TOF method, and using the experimental values ​​obtained for the mass-to-charge ratio of the peaks in the spectrum corresponding to the ions of the so-called reference protein of the calibration composition as calibration values. d) A step of calculating the calibration relationship based on the theoretical mass-to-charge ratio and calibration value of the reference protein ions. Methods that include...

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