CN108593753B

CN108593753B - Internal standard correction method for detecting microorganisms through internal standard substance spectrum

Info

Publication number: CN108593753B
Application number: CN201810346086.1A
Authority: CN
Inventors: 战晓微; 马庆伟; 宋召; 谭甜霞
Original assignee: Beijing Clin Bochuang Biotechnology Co Ltd
Current assignee: Beijing Clin Bochuang Biotechnology Co Ltd
Priority date: 2016-11-25
Filing date: 2016-11-25
Publication date: 2020-06-05
Anticipated expiration: 2036-11-25
Also published as: CN107024530B; CN108519267B; CN108802162A; CN107024530A; CN108593753A; CN108519267A; CN108802162B

Abstract

The present invention provides a method for mass spectrometric detection of microorganisms by means of internal standards having known molecular weights and mass to charge ratios which do not interfere with the peak profiles of the characteristic proteins of the microorganisms during their detection. The invention also provides the internal standard composition, the reagent composition and a related detection product thereof. The method for correcting the detection of a single microorganism sample by using the internal standard substance can be suitable for various microorganism samples including fungi, bacteria and the like for MALDI-TOF MS detection, improves the accuracy of microorganism identification, and simultaneously makes up the defect that only the external standard substance is used for correction in the current market.

Description

Internal standard correction method for detecting microorganisms through internal standard substance spectrum

Technical Field

The invention belongs to the technical field of biology, and relates to internal standard correction for detecting a microorganism sample by a time-of-flight mass spectrometry system.

Background

Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) technology is one of proteomics and genomics technologies which are rapidly developed in recent years, has the characteristics of high sensitivity, high accuracy, high resolution and the like, provides a rapid and high-flux analysis and test means for life science research, early warning and auxiliary diagnosis of clinical major diseases and the like, and is a good method for identifying pathogenic microorganisms.

The principle of MALDI-TOF-MS is a process in which a laser is used to irradiate a co-crystallized thin film formed by a sample and a matrix, which absorbs energy from the laser to transfer to biomolecules, and in which protons are transferred to or obtained from the biomolecules during ionization, thereby ionizing the biomolecules. The principle of TOF is that ions are accelerated to fly through a flight tube under the action of an electric field, and are detected according to different flight times of arriving at a detector, namely, the mass-to-charge ratio (M/Z) of the ions is measured to be in direct proportion to the flight time of the ions, and the ions are detected. Although the accuracy of MALDI-TOF-MS is as high as 0.1% -0.01%, the accuracy is far higher than that of SDS electrophoresis and high performance gel chromatography which are conventionally applied at present. The MALDI-TOF MS technology has the characteristics of rapidness, accuracy, stability, high flux, low cost and the like; MALDI-TOF MS technology is the routine rapid analysis technology of clinical microorganism, also is one of the analytical techniques of resistant microorganism; the MALDI-TOF MS technology provides effective identification technical support for the construction of a pathogenic bacteria resource library. However, because factors such as system errors affect the accurate quantification of experimental results, when a sample is detected by mass spectrometry, an internal standard with known concentration and proportion is introduced, so that the accuracy and precision of single microorganism sample identification are improved.

The existing curve fitting method is widely applied at home and abroad, and Jiangyue Wu and the like (anal. chem.1997,69, 3767-; when Mazarin et al (anal. chem. 2006,78, 2758-; in 2009, Lv Shuang et al successfully developed a MALDI-TOF-MS quantitative method of phosphorylated peptide by using a fitting curve.

The technology is characterized in that acidic matrix is added into complete microorganisms to assist cell lysis, cell lysate (small protein or polypeptide) is excited by laser to form a peptide mass fingerprint spectrum, and the peptide mass fingerprint spectrum is compared and analyzed with a constructed generic and species level common reference spectrum library, so that identification of the microorganisms is realized. And the detection of various characteristic peptides or peptide fingerprints is identified by a series of peak curves with specific mass-to-charge ratios (M/Z). The accuracy of sample collection by using a MALDI-TOF MS mass spectrometer is a key factor of a protein fingerprint spectrum microorganism identification system, and if a peak value curve of a detection result has a certain degree of error (such as peak value shift of 1-10 Da molecular weight) due to the influence of impurities of cell lysate or system errors (continuous supplement) in the detection process, the correction by a standard substance is generally needed so that the measured peak value curve is matched with an actual peak value curve.

Yuan Xianglin et al (report on analytical chemistry research, 29 st volume 1 of 2001) reported that in matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the quantitative analysis of ginsenoside Rg3, rutin was selected as an internal standard, which was superior to raffinose in terms of reproducibility and linearity. The improvement of the resolution and the representation of the concentration of the substance to be measured by the relative peak area can obviously reduce the average relative error and improve the quantitative result. However, this method is not used in mass spectrometric detection of microorganisms, and is different from the linear mode used in MALDI-TOF MS detection of microorganisms, in which the quantitative analysis of ginsenoside Rg3 uses a reflectance mode, and the detection range of the sample to be detected is less than 1000Da, and the detection interval is limited to 400-900(M/Z), which cannot satisfy the detection interval of microorganisms, and thus is limited.

Chinese patent application 2014100902157, the name of the invention "polypeptide standard substance for early diabetes diagnosis" discloses an internal standard polypeptide for mass spectrum detection of early diabetes, which is derived from human serum albumin and has 19 amino acid sequences. The incubation time with a longer period is needed, meanwhile, the internal standard peptide is used for participating in result judgment, the risk possibility of suffering from diabetes is judged by calculating the ratio of the target peptide to the internal standard peptide, and whether the detection result of the sample is accurate or not is not judged. However, this method is not used for MALDI-TOF MS detection of microorganisms, and the detection range is less than 1000Da, which is much different from the detection range of microorganisms 2000Da-20000Da, and cannot satisfy the detection range of microorganisms, so it is limited.

As the closest prior art, chinese patent application 201510246677.8, entitled "mass spectrometer molecular weight calibration standard for microorganism identification and preparation method and application thereof" discloses a group of using 18 escherichia coli characteristic proteins with mass-to-charge ratios m/z of 2094, 2466, 3149, 4364, etc. respectively, as standards for calibrating characteristic spectra and stabilizing calibration effects. However, the standard substance involved in the present invention can effectively detect microorganisms, but it must be subjected to parallel detection before mass spectrometry detection to perform molecular weight correction on a mass spectrometer and cannot directly determine the accuracy of the detection result of a single sample, and still belongs to an external standard substance, and steps are added in the detection process, which is not favorable for high-throughput, rapid and convenient detection of a large number of microorganism samples. In summary, none of the mass spectrometry samples reported so far can satisfy the correction of single sample detection in identifying a microorganism sample, wherein the MALDI-TOF MS method only uses a method of adding an internal standard for quantitative determination, and no report is made on the identification of a microorganism by the MALDI-TOF MS method.

At present, in the process of detecting microorganisms by using a MALDI-TOF-MS method on the market, the detection range is mainly 2000-20000Da (Calderaro et al), although the mass spectrum peak of 400-20000 Da (Pignone et al, Shah et al, Kumar et al, Edwards-Jones et al) is reported to be used for the research of identification and typing, the characteristic peak in the mass range is not used for the identification of a MALDI-TOF-MS instrument on the market. In the 2000-20000Da detection range, the main characteristic peak of the microorganism is less than 12000Da (Winkler, etc.), so in the existing research of detecting the microorganism by laser mass spectrometry, the external standard, even a plurality of external standards are still used for correction, thereby increasing the detection time and the detection cost. Therefore, there is a need for a new method for detecting proteins characteristic of microorganisms by a time-of-flight mass spectrometry system.

Disclosure of Invention

The principle of the invention is as follows: according to the prior MALDI-TOF-MS detection of microorganism mass-to-charge ratio interval (3000-13000m/z), it was first proposed to use a single internal standard of less than 3000m/z or greater than 12000m/z, which has a known molecular weight and mass-to-charge ratio, which does not interfere with the peak profile of the microorganism-characteristic protein during the detection of the microorganism. Therefore, the internal standard substance is added into each microorganism sample to be detected, so that a specific mass spectrogram is generated simultaneously with each microorganism sample, and the mass spectrogram of each microorganism sample is corrected through the known molecular weight of the standard substance and the corresponding characteristic peak thereof (for example, the molecular weight measured value of the characteristic protein of the microorganism to be detected is corrected according to the difference value of the molecular weight to be detected and the actual molecular weight of the standard substance, and if the actual measured value is corrected according to the difference value), so that the accuracy and precision of identification of a single microorganism sample are improved. As the internal standard substance selected by the invention for correction is selected at 1000-3000Da or 13000-20000Da, the detection effect can be achieved, and on the basis of not influencing the identification of microorganisms, the added internal standard substance can correct the whole spectrogram of a single sample so as to make up the defect that MALDI-TOF MS can cause deviation in detection.

Accordingly, it is a first object of the present invention to provide a method for the detection of microorganisms by time-of-flight mass spectrometry with an internal standard, the method comprising:

(1) pretreating a microorganism sample;

(2) adding an internal standard of 1000Da < average molecular weight <3000Da or 12000Da < average molecular weight <20000Da to the pre-treated microbial sample;

(3) carrying out mass spectrum detection on the microorganism sample containing the internal standard substance, so that the microorganism sample and the internal standard substance simultaneously generate a specific mass spectrogram, and correcting the mass spectrogram of each microorganism sample through the known molecular weight of the standard substance and a characteristic peak corresponding to the known molecular weight, thereby obtaining an accurate detection result;

wherein the internal standard may be a polypeptide or/and protein standard or a combination thereof known within the above average molecular weight.

In one embodiment, the internal standard is selected from 1000Da<Average molecular weight<2000Da, or a combination thereof. In a specific embodiment, the internal standard is selected from the group consisting of polypeptide P (33507-63-0), molecular weight 1347.63Da, and sequence SEQ ID NO: 1: RPKPQQFFGLM-NH 2; or, is selected from the polypeptide P₁₄(synthetic peptide) with a molecular weight of 1533.85Da and a sequence of SEQ ID NO: PPPPPPPPPPPPPPR are provided.

In one embodiment, the internal standard is selected from known polypeptide or/and protein standards of 2000Da < average molecular weight <3000Da or combinations thereof. In a specific embodiment, the internal standard is selected from the polypeptides ACTHfragment18-39(human), molecular weight 2465.19Da, and sequence SEQ ID NO: 3: RPVKVYPNGAEDESAEAFPLEF are provided.

In one embodiment, the internal standard is selected from the group consisting of 12000Da < known polypeptide or/and protein standards of average molecular weight <180000Da or combinations thereof. In a specific embodiment, the internal standard is selected from horse myoglobin apomyoglobin (equine), has a molecular weight of 169952 Da and has the sequence of SEQ ID NO: 4: GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASE DLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPG DFGADAQGAMTKALELFRNDIAAKYKELGFQG are provided.

In still other embodiments, the internal standard is selected from any combination of the average molecular weight ranges described above. In a specific embodiment, wherein the internal standard is selected from the group consisting of polypeptide substance P (33507-63-0) in combination with equine myoglobin Apomyoglobin (equine). In another specific embodiment, the internal standard is selected from P₁₄A combination of r (synthetic peptide) and apomyoglobin (equine); in other specific embodiments, the internal standard is selected from the group consisting of ACTH fragment18-39(human) in combination with equine myoglobin apomyoglobin (equine); and, in a further specific embodiment, the internal standard is selected from the group consisting of polypeptide substance P (33507-63-0) and ACTH fragment18-39(human) in combination.

It is a second object of the present invention to provide an internal standard for use in the above method.

The third purpose of the invention is to provide a reagent composition for the pretreatment of a time-of-flight mass spectrum microbial sample, which mainly comprises the components I, II, III and α -cyano-4-hydroxycinnamic acid, acetonitrile and formic acid, acetonitrile, trifluoroacetic acid and the internal standard substance.

In one embodiment, where component I comprises 50.0% (v/v) acetonitrile, 35.0% (v/v) formic acid, the balance water component II comprises 52.5% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid, 15mg/ml (m/v) α -cyano-4-hydroxycinnamic acid, the balance water component III comprises polypeptide species P (33507-63-0) (average molecular weight 1347.3 Da).

In one embodiment, where component I comprises 50.0% (v/v) acetonitrile, 35.0% (v/v) formic acid, the balance water component II comprises 52.5% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid, 15mg/ml (m/v) α -cyano-4-hydroxycinnamic acid, the balance water component III comprises P₁₄R (synthetic peptide) (average molecular weight 1533.85 Da).

In one embodiment, where component I comprises 50.0% (v/v) acetonitrile, 35.0% (v/v) formic acid, the balance water component II comprises 52.5% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid, 15mg/ml (m/v) α -cyano-4-hydroxycinnamic acid, the balance water component III comprises ACTH fragment18-39(human) (average molecular weight 2465.19 Da).

In one embodiment, where component I comprises 50.0% (v/v) acetonitrile, 35.0% (v/v) formic acid, the balance water component II comprises 52.5% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid, 15mg/ml (m/v) α -cyano-4-hydroxycinnamic acid, the balance water component III comprises equine myoglobin Apomyoglobin (equine) (average molecular weight 1699 Da).

In one embodiment, where component I comprises 50.0% (v/v) acetonitrile, 35.0% (v/v) formic acid, balance water component II comprises 52.5% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid, 15mg/ml (m/v) α -cyano-4-hydroxycinnamic acid, balance water component III comprises polypeptide species P (33507-63-0) (average molecular weight 1347.3Da), equine myoglobin Apomyoglobin (equine) (average molecular weight 169952 Da).

In one embodiment, where component I comprises 50.0% (v/v) acetonitrile, 35.0% (v/v) formic acid, the balance water component II comprises 52.5% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid, 15mg/ml (m/v) α -cyano-4-hydroxycinnamic acid, the balance water component III comprises P₁₄R (synthetic peptide) (average molecular weight 1533.85Da), equine myoglobin (equine) (average molecular weight 169952 Da).

In one embodiment, where component I comprises 50.0% (v/v) acetonitrile, 35.0% (v/v) formic acid, the balance water component II comprises 52.5% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid, 15mg/ml (m/v) α -cyano-4-hydroxycinnamic acid, the balance water component III comprises ACTH fragment18-39(human) (average molecular weight 2465.19Da), equine myoglobin Apomyoglobin (1699) (average molecular weight 16910 Da).

In one embodiment, where component I comprises 50.0% (v/v) acetonitrile, 35.0% (v/v) formic acid, the balance water component II comprises 52.5% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid, 15mg/ml (m/v) α -cyano-4-hydroxycinnamic acid, the balance water component III comprises polypeptide species P (33507-63-350) (average molecular weight 1347.3Da), ACTH fragment18-39(human) (average molecular weight 2465.19 Da).

It is a fourth object of the present invention to provide a test product for mass spectrometric identification of unknown microorganisms prepared by the above internal standard or reagent composition.

In one embodiment, the product is a time-of-flight mass spectrometry detection kit for microorganisms comprising:

(1) reagent composition for pretreatment of a microbial sample;

(2) an internal standard composition.

In one embodiment, the kit further comprises: (3) other mass spectrum reagents comprise negative quality control substances and positive quality control substances.

In another embodiment, the kit further comprises target pieces for spotting and mass spectrometry, and software for comparing and correcting the molecular weights of the standard substance and the test substance.

A fifth object of the present invention is to provide an internal standard calibration method for identifying a microbial sample by use of the above internal standard or reagent composition or detection product in a time-of-flight mass spectrometry system, comprising the steps of:

(1) selecting a part of appropriate single colony by using a sterilized toothpick (or 1 mul inoculating loop) and placing the single colony in a centrifuge tube filled with 10 mul of the component I for cell disruption, releasing protein and polypeptide and cracking for 5 minutes;

(2) adding 1 μ l of the above solution to the hole of the target plate, and drying at room temperature;

(3) covering the same hole with 1 μ l of internal standard substance, and naturally drying;

(4) opening the microtube of the component II, transferring 1 mul of the component II to the same hole site, and naturally drying;

(5) and (3) putting the target plate into a time-of-flight mass spectrometry system for detection, wherein the molecular weight of the characteristic protein of the microorganism to be detected is corrected by comparing the difference value between the molecular weight to be detected of the internal standard substance and the actual molecular weight.

In one embodiment, the microorganism detection is the determination of the genus, species, subspecies or subtype of microorganism. In a particular embodiment, the detection of microorganisms is of a non-diagnostic purpose, detecting pathogenic bacteria, contaminating bacteria, drug-resistant bacteria, etc., or pathogenic bacteria with a diagnostic purpose.

In one embodiment, the microorganism in any of the above embodiments is a microorganism in environmental pollution, a microorganism in food quarantine, a microorganism in import and export commodities, a drug-resistant microorganism in pharmaceutical research, or the like.

In one embodiment, the microorganism of any of the above embodiments is a prokaryotic microorganism, a eukaryotic microorganism. In a specific embodiment, the prokaryotic microorganism comprises a bacterium. The eukaryotic microorganism is fungi including yeast, mold, etc.

Principles and definitions

The principle of identifying the microorganism by the time-of-flight mass spectrometry system is as follows: different microorganisms have different proteins, after being processed, microorganism samples are detected by a time-of-flight mass spectrometry system, different microorganisms have different characteristic fingerprint spectrums, and different microorganisms can be distinguished by comparing the characteristic fingerprint spectrums of the known microorganisms in a database through time-of-flight mass spectrometry system analysis software, namely, the identification result of the microorganisms is given. Because the characteristic fingerprint spectrogram of the microorganism is the only judgment basis for identifying the microorganism by the time-of-flight mass spectrometry system, the accuracy of the spectrogram is the premise of correct microorganism identification. And adding a standard substance into each single sample to be detected, correcting the single sample through analysis software, and then carrying out microorganism identification, thereby improving the accuracy of single sample detection.

It should be noted that although the present invention can be used for detecting microorganisms, the present invention merely corrects the detection result so that the detection result coincides with the actual result. The detection method of the invention does not belong to disease diagnosis or detection, because the detection of microorganisms by a time-of-flight mass spectrometry system requires the preparation of a characteristic protein map of the microorganism to be detected in advance, and the detection cannot be completed only by the internal standard substance of the invention.

Technical effects

(1) According to the invention, through research on adding standard substances into the microbial samples, the fact that the addition of the internal standard substances can correct a single microbial sample and is used together with a microbial sample processing reagent of a time-of-flight mass spectrometry system is found, so that the accuracy of microbial identification can be improved, and the defect that only external standard substances are used for correction in the current market is overcome;

(2) the molecular weight range of the added internal standard substance is outside the microorganism identification range, and the requirements of detection and correction can be met within the detection range;

(3) the method for correcting the detection of a single microorganism sample by using the internal standard substance can be suitable for various microorganism samples for MALDI-TOF MS detection, such as fungi, bacteria and the like.

Drawings

FIG. 1-1: citrobacter koseri, Citrobacter koseri; FIGS. 1-2: escherichia coli

FIGS. 1 to 3: enterococcus faecalis; FIGS. 1 to 4: klebsiella pneumoniae

FIGS. 1 to 5: stenotrophomonas maltophilia

FIGS. 1 to 6: pseudomonas aeruginosa; FIGS. 1 to 7: salmonella sp.

FIGS. 1 to 8: klebsiella oxytoca; FIGS. 1 to 9: human staphylococcus staphyloccoccushommins

FIGS. 1 to 10: acinetobacter baumannii

FIGS. 1 to 11: enterobacter cloacae; FIGS. 1 to 12: serratia marcescens Serratiamarcescens

FIG. 2-1: polypeptide mass spectrogram dissolved by trifluoroacetic acid/acetonitrile organic solvent

FIG. 2-2: dissolving the polypeptide mass spectrogram by using ultrapure water; FIGS. 2 to 3: dissolution of internal standard compositions with ultrapure water

FIGS. 2 to 4: dissolution of internal standard compositions using trifluoroacetic acid/acetonitrile

FIG. 3-1: adding internal standard and treating microorganism sample Escherichia coli (ATCC8739) by using plate-smearing method

FIG. 3-2: adding internal standard, and treating microorganism sample Escherichia coli (ATCC8739) by one-step extraction method

FIGS. 3-3: addition of internal standard microbial samples of Escherichia coli (ATCC8739) were treated by three-step centrifugation

FIGS. 3-4: mixing and adding the internal standard composition (drawing open the internal standard); FIGS. 3 to 5: composition diagram with mixed addition of internal standard

FIG. 4-1: addition of internal standard and smear treatment of microbial samples E.coli (ATCC8739) with biased peak profiles

FIG. 4-2: adding internal standard, treating microorganism sample by using smearing method, and correcting peak pattern of Escherichia coli (ATCC8739) after deviation

FIG. 5: m/z1351 has obvious characteristic peak pattern

FIG. 6-1: staphylococcus haemolyticus; FIG. 6-2: staphylococcus aureus bacteria; FIGS. 6-3: human staphylococcus

FIGS. 6 to 4: pseudomonas aeruginosa; FIGS. 6 to 5: proteus vulgaris; FIGS. 6 to 6: acid-producing Klebsiella sp

FIGS. 6 to 7: acinetobacter baumannii; FIGS. 6 to 8: streptococcus agalactiae; FIGS. 6 to 9: enterococcus faecium

FIG. 7: staphylococcus epidermidis; FIG. 8: aeromonas hydrophila

FIG. 9: protein fingerprint spectrum for identifying escherichia coli by using ultrapure water dissolution of equine myoglobin standard substance

Detailed Description

In order to further understand the technical features of the present invention, the present invention is described in detail with reference to the specific embodiments below. The embodiments are given by way of illustration only and not by way of limitation, and the scope of the invention should be determined by that of the claims which follow and that of insubstantial modifications made by those skilled in the art based on the teachings of the invention.

The first embodiment is as follows: microorganism identification by using time-of-flight mass spectrometry system microorganism sample pretreatment kit

Strain culture and pretreatment: culturing multiple clinical isolated strains stored in a clinical laboratory of Beijing Hospital at 37 ℃ for 24 hours to obtain corresponding single colonies, picking partial appropriate single colonies with a sterilizing toothpick (or 1 mul inoculating loop), coating the single colonies on a corresponding point of a target plate, sucking 1 mul of component I to cover the point, naturally drying, sucking 1 mul of component II to cover the same hole site, naturally drying, detecting on a machine, identifying and analyzing, wherein the identification results are shown in table 1 and figures 1-1 to 1-12.

TABLE 1 identification results of 5 strains of bacteria in example I

From the above identification results, the main characteristic peaks in the mass spectrogram of the microorganism (see fig. 1-1 to fig. 1-12) are distributed between 2000 and 13000Da, so that the mass spectrogram peaks similar to the characteristic peaks of the microorganism cannot be generated when the molecular weight of the internal standard substance added in the invention is less than 2000Da or/and more than 13000Da, thereby theoretically ensuring the feasibility and the accuracy of microorganism identification.

EXAMPLE two preparation of an internal reference composition for identification of microorganisms by time-of-flight mass spectrometry System

(1) Dissolving standard substance by using trifluoroacetic acid/acetonitrile

Dissolving polypeptide standard substance dry powder (namely polypeptide P (33507-63-0) with trifluoroacetic acid/acetonitrile organic solvent (0.1% trifluoroacetic acid and 10% acetonitrile) into 100fmol/ul solution, carefully mixing the solution, sucking 1 mul of the solution to cover a target plate point, naturally drying the solution, sucking 1 mul of component II (or other matrix solution used in microorganism identification) in a microorganism sample processing reagent of a commercial time-of-flight mass spectrometry system to cover the same point, naturally drying the solution, and detecting by a time-of-flight mass spectrometer, wherein after the detection result of the standard substance is corrected by an instrument, the detection result of the standard substance is shown in figure 2-1, and the standard substance has an obvious characteristic peak at m/z1348.76, which shows that the standard substance can be dissolved by trifluoroacetic acid/acetonitrile and has good detection effect.

(2) Dissolving standard substance with ultrapure water

Dissolving a polypeptide standard product dry powder (namely polypeptide P (33507-63-0) with the molecular weight of 1347Da) into a solution with the concentration of 500fmol/ul by using ultrapure water, carefully mixing the solution uniformly, sucking 1 mul of the solution to cover the point of a target plate, naturally drying the solution, sucking 1 mul of component II (or other matrix liquid used in microorganism identification) in a commercial time-of-flight mass spectrometry system microorganism sample processing reagent to cover the same point, naturally drying the solution, and detecting by using a time-of-flight mass spectrometer, wherein a detection result is shown in a figure 2-2, and the detection result has an obvious characteristic peak at a position of m/z1348.88, which shows that the standard substance can be dissolved by using the ultrapure water and has a good detection effect.

(3) Dissolution of internal standard compositions with ultrapure water

A mixed solution of a polypeptide substance (i.e., polypeptide P (33507-63-0), molecular weight 1347Da) at a final concentration of 500 fmol/. mu.L and P14R (molecular weight 1533) at a final concentration of 50 fmol/. mu.L was prepared with ultrapure water. After carefully mixing, sucking 1 mul to cover the target plate point, naturally drying, sucking 1 mul of component II (or other matrix liquid used in microorganism identification) in the microorganism sample processing reagent of the commercial time-of-flight mass spectrometry system to cover the same point, after naturally drying, detecting by a time-of-flight mass spectrometer, and after instrument correction, the detection result of the internal standard composition is shown in figure 2-3, wherein the internal standard composition has obvious characteristic peaks at m/z 1348.64 and m/z1534.65, which shows that the internal standard composition can be dissolved by ultrapure water and has good detection effect.

(4) Dissolution of internal standard compositions using trifluoroacetic acid/acetonitrile

A mixed solution of a polypeptide substance (molecular weight 1347Da) at a final concentration of 500 fmol/. mu.L and P14R (molecular weight 1533) at a final concentration of 50 fmol/. mu.L was prepared using a trifluoroacetic acid/acetonitrile organic solvent (0.1% trifluoroacetic acid and 10% acetonitrile). After carefully mixing, sucking 1 mul to cover the target plate point, naturally drying, sucking 1 mul of component II (or other matrix liquid used in microorganism identification) in the microorganism sample processing reagent of the commercial time-of-flight mass spectrometry system to cover the same point, and after naturally drying, detecting by a time-of-flight mass spectrometer. After the calibration of the instrument, the detection results of the internal standard composition are shown in FIGS. 2-4, and the internal standard composition has obvious characteristic peaks at m/z 1348.48 and m/z1534.48, which indicates that the internal standard composition can be dissolved by trifluoroacetic acid/acetonitrile organic solvent and has good detection effect.

As shown in the above figure, the theoretical molecular weight (1347.63Da) of the internal standard polypeptide P (33507-63-0) and the theoretical molecular weight (1533.85Da) of P14R have slight differences from the actually detected molecular weight, and each detection value of the same internal standard (such as the polypeptide P) has a certain difference, which indicates that a systematic error is inevitable in the process of detecting the molecular weight of the microbial characteristic protein by laser mass spectrometry. Therefore, in the actual process of detecting the microorganisms, the single or combined internal standard substance is added to correct the system error, so that the characteristic proteins of different microorganisms and similar molecular weights thereof can be detected and distinguished more accurately.

Example optimization of internal standard composition addition method for identifying microorganisms in a time-of-flight mass spectrometry System

(1) Direct addition internal standard compositions

Treating a microorganism sample escherichia coli (ATCC8739) by using a smearing method, and directly adding an internal standard composition to a sample point, wherein the specific operation method comprises the following steps: picking single bacterial colony of escherichia coli by using an aseptic toothpick, uniformly smearing the single bacterial colony on a target plate point, naturally drying, covering 1 mu l of an internal standard composition on the same point (or covering 1 mu l of a component I in a commercial time-of-flight mass spectrometry system microorganism sample processing reagent on a sample point, and covering 1 mu l of the internal standard composition after natural drying), naturally drying, sucking a component II (or other matrix liquid used for microorganism identification) in the commercial time-of-flight mass spectrometry system microorganism sample processing reagent of 1 mu l, covering the same point, and naturally drying and then carrying out time-of-flight mass spectrometry detection. The results of this sample are shown in FIG. 3-1, which shows a distinct peak at m/z1348, indicating that the internal standard composition can be directly added to the sample spot for detection when the microplate method is used to process the microbial sample.

The method comprises the following steps of (1) processing microorganism sample escherichia coli (ATCC8739) by using a one-step extraction method, and directly adding an internal standard composition to a sample point, wherein the specific operation method comprises the following steps: adding 30 mul of component I in a commercial time-of-flight mass spectrometry system microorganism sample processing reagent into a 200 mul centrifuge tube, picking a single colony in an upper tube by using 1 mul of sterile inoculating loop or a gun head, shaking and uniformly mixing for 5min, adding 1 mul of sample to a target plate, naturally drying, covering 1 mul of internal standard composition on the same point, naturally drying, absorbing 1 mul of component II (or other matrix liquid used in microorganism identification) in the commercial time-of-flight mass spectrometry system microorganism sample processing reagent to cover the same point, and detecting the time-of-flight mass spectrometer after natural drying. After the sample is corrected by the instrument, the detection result of the sample is shown in a figure 3-2, and a characteristic peak is obvious at m/z1348, which indicates that when the microbial sample is processed by using a further extraction method, the internal standard composition can be directly added into a sample point for detection.

The method comprises the following steps of (1) processing microorganism sample escherichia coli (ATCC8739) by using a three-step centrifugation method, and directly adding an internal standard composition to a sample point, wherein the specific operation method comprises the following steps: adding 300 mu l of pure water into a 1.5ml or 2ml centrifuge tube, inoculating and taking a single escherichia coli colony in an upper tube, uniformly oscillating, adding 900 mu l of ethanol, oscillating, centrifuging at 12000rpm for 2 minutes, discarding supernatant, centrifuging for 2 minutes, naturally drying, adding 50 mu l of 70% formic acid, sufficiently oscillating, adding 50 mu l of acetonitrile, sufficiently oscillating, centrifuging at 12000rpm for 2 minutes, adding 1 mu l of sample onto a target plate, naturally drying, covering 1 mu l of internal standard composition on the same point, naturally drying, sucking 1 mu l of component II (or other matrix liquid used in microorganism identification) in a commercial time-of-flight mass spectrometry system microorganism sample processing reagent to cover the same point, and naturally drying and then carrying out time-of-flight mass spectrometer detection. The results of this sample are shown in FIGS. 3-3, which show a distinct characteristic peak at m/z1348, indicating that the internal standard composition can be directly added to the sample spot for detection when the microbial sample is processed by a three-step centrifugation method.

(2) Mixed addition internal standard composition

Treating microorganism sample escherichia coli (ATCC8739) by using a smearing method, mixing an internal standard composition and a matrix solution according to a ratio of 1:1, and adding the mixture to a sample point, wherein the specific operation method comprises the following steps: picking single bacterial colony of escherichia coli by using an aseptic toothpick, uniformly coating the single bacterial colony on a target plate point, naturally drying, covering a mixture of 1 mu l of an internal standard composition and a component II (or other matrix liquid used for microorganism identification) in a microorganism sample processing reagent of a commercial flight time mass spectrometry system on the same point, naturally drying, and then carrying out flight time mass spectrometry detection. The results of this sample are shown in FIGS. 3-4 and 3-5, which show distinct characteristic peaks at m/z1348 and m/z1534, after calibration by the instrument, indicating that the internal standard composition can be mixed with the matrix solution and added to the sample spot for detection when the microbial sample is processed by the spatula.

Example four evaluation of the Effect of correction of internal composition for identification of microorganisms by time-of-flight Mass Spectrometry System

An internal standard composition is utilized to correct a mass spectrogram of a time-of-flight mass spectrometry system for detecting microorganisms, escherichia coli (ATCC8739) is inoculated on a Columbia blood agar culture medium, the mixture is cultured for 24 hours at 37 ℃, an aseptic toothpick picks up a single escherichia coli colony, the single escherichia coli colony is uniformly coated on a target plate point, after natural drying, 1 mul of the internal standard composition is covered on the same point (or 1 mul of component I in a commercial time-of-flight mass spectrometry system microorganism sample processing reagent is covered on the sample point, 1 mul of the internal standard composition is covered after natural drying), natural drying is carried out, 1 mul of component II (or other matrix liquid used for microorganism identification) in the commercial time-of-flight mass spectrometry system microorganism sample processing reagent is sucked and covered on the same point, after natural drying, time mass spectrometry detection is carried out, the detection result of the sample is shown as figure 4-1, and no obvious characteristic peak exists at the position of m/z1348, and (4) showing that the mass spectrum peak in the mass spectrogram has deviation, the mass spectrum detection result of the sample is not credible, and the instrument correction and data acquisition are required to be carried out again.

The escherichia coli sample is judged by the characteristic peak m/z1348 value of the internal standard composition, the mass spectrogram deviates, mass spectrum data are collected after the instrument is corrected again, the detection result is shown as a figure 4-2, and the m/z1348 position in the mass spectrogram has an obvious characteristic peak, which indicates that the mass spectrum peak of the mass spectrogram does not deviate, and the mass spectrum detection result of the sample is credible.

Example five evaluation of the Effect of internal composition correction for identification of microorganisms by time-of-flight Mass Spectrometry System

An internal standard composition is utilized to correct a mass spectrogram of a time-of-flight mass spectrometry system for detecting microorganisms, escherichia coli (ATCC8739) is inoculated on a Columbia blood agar culture medium, the mixture is cultured for 24 hours at 37 ℃, an aseptic toothpick picks up a single escherichia coli colony, the single escherichia coli colony is uniformly coated on a target plate point, after natural drying, 1 mul of the internal standard composition is covered on the same point (or 1 mul of component I in a commercial time-of-flight mass spectrometry system microorganism sample processing reagent is covered on the sample point, 1 mul of the internal standard composition is covered after natural drying), natural drying is carried out, 1 mul of component II (or other matrix liquid used for microorganism identification) in the commercial time-of-flight mass spectrometry system microorganism sample processing reagent is absorbed and covered on the same point, after natural drying, time mass spectrometry detection is carried out, the detection result of the sample is shown in figure 5, and the sample has an obvious characteristic peak at the position of m/z1351, and no obvious characteristic peak exists at m/z1348, which indicates that the mass spectrum peak in the mass spectrum has deviation, the data is identified by identification software with a correction function, the identification result is Escherichia coli, and the corresponding credibility score is 94, which indicates that the internal standard has good correction effect.

EXAMPLE six detection of microbial samples Using time-of-flight Mass Spectrometry System microbial sample treatment reagents and internal Standard compositions of the invention

Clinical common pathogenic bacteria (table 2) are detected according to the method described in the fifth embodiment, the detection results are shown in fig. 6-1 to 6-9, and the identification results after software analysis and treatment are shown in table 3.

TABLE 2 List of clinically common pathogenic bacteria

Strain numbering	Latin name of strain	Chinese name of strain
			6‐1	Staphylococcus haemolyticus	Hemolytic staphylococcus
6‐2	Staphylococcus aureus	Staphylococcus aureus
			6‐3	Staphylococcus hominis	Human staphylococcus
6‐4	Pseudomonas aeruginosa	Pseudomonas aeruginosa
			6‐5	Proteus vulgaris	Proteus vulgaris
6‐6	Klebsiella oxytoca	Acid-producing Klebsiella sp
			6‐7	Acinetobacter baumannii	Acinetobacter baumannii
6‐8	Streptococcus agalactiae	Streptococcus agalactiae
			6‐9	Enterococcus faecium	Enterococcus faecium

TABLE 3 identification results of clinically common pathogenic bacteria

EXAMPLE seven detection of pathogenic bacteria in patients with skin infections Using time of flight Mass Spectrometry System microbial sample treatment reagents and internal Standard compositions of the invention

And (3) inoculating and culturing a suppurative secretion sample at the wound of a patient infected by a surgical site in a certain hospital to obtain a single bacterium of suspected pathogenic bacteria, treating the microorganism sample by using the method of the fifth embodiment, detecting by using a time-of-flight mass spectrometer to obtain a detection result as shown in figure 7, wherein the identification result is that the result of the staphylococcus epidermidis is consistent with the identification result of a full-automatic biochemical identification instrument.

EXAMPLE eight detection of pathogenic bacteria in food Using time-of-flight Mass Spectrometry System microbial sample treatment reagent and internal Standard composition of the invention

The mass spectrometric identification results are shown in FIG. 8.

EXAMPLE nine identification of Escherichia coli Using equine myoglobin Standard substance

The method comprises the following steps: the ultra-pure water is dissolved, and the mass spectrum identification result is shown in figure 9.

Sequence listing

<110> Beijing resolute Xinbo Chuang Biotech Co., Ltd

<120> method for detecting microorganism by internal standard substance spectrum and product thereof

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>11

<212>PRT

<213> microorganism (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400>1

Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met

1 5 10

<210>2

<211>15

<212>PRT

<213> microorganism (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400>2

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Arg

15 10 15

<210>3

<211>22

<212>PRT

<213> microorganism (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400>3

Arg Pro Val Lys Val Tyr Pro Asn Gly Ala Glu Asp Glu Ser Ala Glu

1 5 10 15

Ala Phe Pro Leu Glu Phe

20

<210>4

<211>153

<212>PRT

<213> microorganism (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400>4

Gly Leu Ser Asp Gly Glu Trp Gln Gln Val Leu Asn Val Trp Gly Lys

1 5 10 15

Val Glu Ala Asp Ile Ala Gly His Gly Gln Glu Val Leu Ile Arg Leu

20 25 30

Phe Thr Gly His Pro Glu Thr Leu Glu Lys Phe Asp Lys Phe Lys His

35 40 45

Leu Lys Thr Glu Ala Glu Met Lys Ala Ser Glu Asp Leu Lys Lys His

50 55 60

Gly Thr Val Val Leu Thr Ala Leu Gly Gly Ile Leu Lys Lys Lys Gly

65 7075 80

His His Glu Ala Glu Leu Lys Pro Leu Ala Gln Ser His Ala Thr Lys

85 90 95

His Lys Ile Pro Ile Lys Tyr Leu Glu Phe Ile Ser Asp Ala Ile Ile

100 105 110

His Val Leu His Ser Lys His Pro Gly Asp Phe Gly Ala Asp Ala Gln

115 120 125

Gly Ala Met Thr Lys Ala Leu Glu Leu Phe Arg Asn Asp Ile Ala Ala

130 135 140

Lys Tyr Lys Glu Leu Gly Phe Gln Gly

145 150

Claims

1. An internal standard calibration method for identifying a microbial sample by a reagent composition for use in a time-of-flight mass spectrometry system, comprising the steps of:

(1) selecting a part of appropriate single colony by using a sterilized toothpick, putting the single colony into a centrifugal tube filled with 10 mul of the component I, carrying out cell disruption, releasing protein and polypeptide, and cracking for 5 minutes;

(2) adding 1 μ l of the above lysis solution to the hole of the target plate, and drying at room temperature;

(3) covering the same hole with 1 μ l of protein standard, and naturally drying;

(5) the target plate is placed in a time-of-flight mass spectrometry system for detection, wherein the molecular weight of the characteristic protein of the microorganism to be detected is corrected by comparing the difference value between the molecular weight to be detected of the internal standard substance and the actual molecular weight;

wherein the main components of the reagent composition comprise acetonitrile and formic acid as a component I, acetonitrile, trifluoroacetic acid and α -cyano-4-hydroxycinnamic acid as a component II, and internal standards with the average molecular weight of 1000Da <3000Da and 12000Da <20000Da as a component III,

wherein the internal standard is selected from polypeptide P (33507-63-0) and/or polypeptide P14R (synthetic peptide) of 1000Da < average molecular weight <2000 Da; and/or the like, and/or,

wherein the internal standard is selected from the polypeptides ACTH fragment18-39(human) of 2000Da < average molecular weight <3000 Da;

and wherein the internal standard is selected from the group consisting of equine apophotoglobin (apomyoglobin) with 12000Da < average molecular weight <180000 Da.

2. The calibration method according to claim 1, wherein the component I comprises acetonitrile of 50.0% by volume, formic acid of 35.0% by volume and the balance water, and the component II comprises acetonitrile of 52.5% by volume, trifluoroacetic acid of 2.5% by volume, α -cyano-4-hydroxycinnamic acid of 15mg/ml and the balance water.

3. The calibration method according to claim 1 or 2, wherein the microorganism detection is determination of genus, species, subspecies or subtype of microorganism; or to detect pathogenic bacteria, contaminating bacteria, drug-resistant bacteria, or pathogenic bacteria with diagnostic purposes in a non-diagnostic destination.

4. The calibration method according to claim 3, wherein the microorganism is a microorganism in environmental pollution, a microorganism in food quarantine, a microorganism in import and export commodities, a drug-resistant microorganism in pharmaceutical research, and the microorganism includes a prokaryotic microorganism and a eukaryotic microorganism.

5. The calibration method of claim 4, wherein the prokaryotic microorganism comprises a bacterium and the eukaryotic microorganism is a fungus comprising a yeast or a mold.