CN106199003A - The construction method in microbial polypeptide mass fingerprint storehouse based on flight time mass spectrum principle - Google Patents
The construction method in microbial polypeptide mass fingerprint storehouse based on flight time mass spectrum principle Download PDFInfo
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- CN106199003A CN106199003A CN201610577592.2A CN201610577592A CN106199003A CN 106199003 A CN106199003 A CN 106199003A CN 201610577592 A CN201610577592 A CN 201610577592A CN 106199003 A CN106199003 A CN 106199003A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
- G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Hematology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Optics & Photonics (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses the construction method in a kind of microbial polypeptide mass fingerprint storehouse based on flight time mass spectrum principle: the bacterial strain of acquisition is carried out pure culture according to its physiological habit, obtains the bacterial strain of logarithmic (log) phase successively；And by morphology, taxonomy, methodology, it is identified；Then the bacterial strain confirmed is carried out pretreatment, use ethanol/formic acid method and 80%TFA extraction method to extract peptide fingerprinting spectrum after drying；And use MALDI TOF MS mass spectrometric platforms to carry out the collection of collection of illustrative plates, process, and then bacterial strain collection of illustrative plates is carried out parameter determination, and the standard spectrogram determined is unified with strain name, mark the banking process of this bacterial strain, complete this bacterial strain builds storehouse；Other bacterial strain banking process the like, complete the structure in microbial polypeptide mass fingerprint storehouse.The invention provides the complete set method from the investigation of bacterial strain to database sharing, this data base's microbe species is complete, will be satisfied with the qualification requirement of microorganism relevant industries.
Description
Technical field
The present invention relates to microbial identification data base, especially relate to a kind of microorganism based on flight time mass spectrum principle
The construction method in peptide mass fingerprinting spectrum storehouse.
Background technology
The microorganisms such as antibacterial, fungus, actinomycetes, rickettsia, mycoplasma, chlamydia, spirillum all contain a large amount of guarantor
The albumen kept, usually ribosomal protein, become relative to microbial metabolic products and other cells easily affected by condition of culture
Point, utilize conservative protein to carry out microbial identification and can ensure that stability and the repeatability of qualification result.So fast and accurately
It is clinical diagnosis, food and disease control inspection that antibacterial, fungus, actinomycetes, mycoplasma, chlamydia, rickettsia etc. carry out qualification
The basic demand of the microbiological art such as survey.
Traditional qualification depends on biochemical identification method, the method except time-consuming, arduously in addition to, to some antibacterial or thin
Flora is difficult to effectively distinguish, and such as part gram-negative bacteria, non-fermented bacillus etc. have inactive biochemical reaction characteristic
Mushroom.Although 16srRNA is the goldstandard of microbial identification, but this technology requires that testing staff's quality is high, and has detection
Cost is high, defect of time-consumingly waiting so long, it is impossible to be widely used in Micro biological Tests.Matrix Assisted Laser Desorption ionization time of flight mass spectrometry
The appearance of (being called for short MALDI-TOF MS) improves the shortcoming that existing authentication method is time-consuming, laborious, cost is high, it is possible to effectively district
Divide the part microbial population that the past is difficult to differentiate between by biochemical identification.
MALDI-TOF MS is a kind of Soft ionization techniques, peptide quality based on MALDI TOF MS
Dactylogram microorganism identification technology is come out the most therewith.This technology is to add acidic matrix auxiliary cell in complete microorganism to split
Solve, laser excitation cell lysate (albumen or polypeptide) formed peptide mass fingerprinting spectrum, and with the genus and species level built
General character reference spectrum compare, thus realize the qualification to unknown microorganism.
At present, market there is two microbial standard peptide fingerprint quality spectrum data storehouse (to be primarily referred to as Brooker company
The SARAMIS data base of Biotyper data base and Mei Liai company), owing to these data bases being used for build standard diagram
The microorganism that the many employings of bacterial strain are external, and there is geographic difference in microorganism, so the part bacterium in this two data base
Strain does not also meet the actual demand of Chinese Clinical, food, diseases monitoring etc., causes China Partial microorganism owing to lacking standard drawing
Compose and the genus level that maybe can only identify can not be identified, bring difficulty to clinical diagnosis and treatment.Such as brucella is in the two
Do not exist in data base, and brucella is a kind of infecting both domestic animals and human pathogenic bacterium the most occurred frequently, the particularly cultivation such as cattle and sheep
In Chang, the frequency of occurrences of this pathogenic bacteria is the highest；Some bacterial strains that altofrequency occurs clinically, such as Acinetobacter bauamnnii, salmonella, list
Increase the strain below portion strain such as Liszt, candidiasis and there is also difficulty in the qualification of kind of level.Micro-in cause of disease based on China
Needs in terms of biological study and the actual demand of the aspect such as clinic, food diagnosis, use strong operability, identify that bacterial strain is accurate
Method build that a microbe species is complete, standard diagram database data amount big and can meet that China is clinical, food diagnosis, disease
The Matrix Assisted Laser Desorption ion mass-spectrometer microbial identification data base of the actual demands such as sick monitoring just seems necessary.
Summary of the invention
Present invention aims to the defect of prior art existence and provide a kind of based on flight time mass spectrum principle
The construction method in microbial polypeptide mass fingerprint storehouse.
For achieving the above object, the present invention can take following technical proposals:
Under the construction method in microbial polypeptide mass fingerprint storehouse based on flight time mass spectrum principle of the present invention includes
State step:
The first step, the standardization of bacterial strain
First the bacterial strain of acquisition is carried out pure training according to respective physiological habit respectively under specific culture medium and condition of culture
Support, obtain the bacterial strain of logarithmic (log) phase successively；And bacterial strain cultivation obtained uses protective agent to carry out preservation so that it is steady in a long-term deposits
Live；
The bacterial strain survived preservation by morphology is verified, it is ensured that the purity of bacterial strain is 100%；By taxonomy to preservation
The bacterial strain of survival is verified, it is ensured that the title of bacterial strain is correct；By distinct methods, same bacterial strain is carried out molecules mirror
Fixed, it is ensured that the concordance of its qualification result；
Second step, builds the extraction of storehouse bacterial strain peptide fingerprinting spectrum
The first step confirms to meet the antibacterial of conditions for building groundwater reservoir, fungus, actinomycetes, mycoplasma, chlamydia, rickettsia bacterial strain enter
Row pretreatment, inactivates thalline and Lipid dissolution, uses ethanol/first acidity extraction peptide fingerprinting spectrum after drying；Will be with
The bacterial strain of spore, the bacterial strain relying on respiratory infectious and biological safety bacterial strain more than two classes use 80%TFA extraction method
Extraction peptide fingerprinting is composed；
3rd step, builds the collection of storehouse bacterial strain peptide fingerprinting spectrum
1) determination of collecting device uses MALDI-TOF MS mass spectrometric platforms to carry out the collection of collection of illustrative plates, and it is as follows that instrument adjusts parameter:
A () ionization laser source excites according to nitrogen, wavelength is 337nm；According to solid-state laser, wavelength is 355nm；
B the acquisition range of the peptide mass fingerprinting spectrum of () microbial standard spectrogram is between 2000-20000Da；
C the electric charge of () laser excitation is the polypeptide with a positive charge or albumen, use linear cation operator scheme, ion source
1 voltage 20 KV, ion source 2 voltage 17-20KV, electron lens voltage 5-10KV, time delay extraction time be 30-400ns it
Between；
D () laser frequency is 40-100HZ；
E () detector voltage, between-2kv to-5kv, with substrate HCCA as background peaks during debugging, determines detector and reaches it
To minimum voltage value during response；
(f) instrument calibration: the laser number of blows gathering spectrogram is set as 40-200 time；Use large intestine bar before data acquisition every time
Bacterium DH5a carries out instrument calibration with the albumen of Myoglobin, the mixing of RNA (ribonucleic acid) enzyme, chooses eight and is uniformly distributed in
Instrument is calibrated by the characteristic protein of 2000-20000Da, and the maximum error of its alignment is less than 150ppm, and Calibration equation is:
y=ax2+ bx+c, wherein y represents m/z, x and represents the time；
2) each target spot of each bacterial strain of acquisition method gathers 3 ~ 5 collection of illustrative plates, and every collection of illustrative plates is by more than 4 times cumulative acquisitions, every time
Accumulated collection of illustrative plates to meet following condition: signal to noise ratio higher than 3.0, baseline is low and flat, main body peak intensity is at 2000-20000
Between, the satisfactory spectrogram of each bacterial strain is at 20 ~ 40；
3) collection of illustrative plates processes and is analyzed in analyzing software by multiple collection of illustrative plates collected, the first characteristic peak to every collection of illustrative plates
Compare, delete the collection of illustrative plates being clearly distinguishable from other collection of illustrative plates on statistical significance, and delete underproof collection of illustrative plates, qualified figure
Spectrum is retained, and collection of illustrative plates number is more than 20, is unsatisfactory for need to again extracting collection, to reach the requirement of Database；
4th step, builds storehouse
1) the bacterial strain collection of illustrative plates of the 3rd step collection is carried out parameter determination
Standard diagram parameter is provided that maximum error 1500-2400ppm at a) single TuPu method peak;B () creates standard peptide
The error of quality fingerprinting reference spectrum is at 200-300ppm；(c) minimum spectrogram frequency: 25%；(d) spectrogram maximum peak number: 70-
80；
2) the standard spectrogram determined is unified with strain name, and mark the banking process of this bacterial strain, complete this bacterial strain
Build storehouse；Other bacterial strain banking process the like, complete the structure in microbial polypeptide mass fingerprint storehouse.
For ensureing the shell-broken effect of thalline, improving the accuracy rate identified, second step extracts building storehouse bacterial strain peptide fingerprinting spectrum
Time, the bacterial strains such as antibacterial, fungus, actinomycetes, mycoplasma, chlamydia, rickettsia are needed to carry out pretreatment, its method is:
The first step, the thalline that picking is appropriate from culture medium, use ultra-pure water to suspend, mix；
Second step, adds dehydrated alcohol, fully mixes, and makes the volume fraction of dehydrated alcohol in mixed liquor reach 70 ~ 80%, stands 1
~ 5 minutes；
3rd step, the mixed solution 10000 ~ 15000rpm/min rotating speed after being stood by second step is centrifuged 3 ~ 5 minutes, goes
Clearly；
4th step, by thalline constant temperature incubation 5 ~ 25 minutes under the conditions of 40 DEG C ± 1 DEG C of precipitation.
Object of the present invention is to provide the complete set method from the investigation of bacterial strain to database sharing, and make with this
For instructing, build the data including the separate sources microorganism fungus kinds such as food, disease control, clinic, industry, agricultural, ocean, environment
Storehouse, this data base's microbe species is complete, not only comprises the type strain of every kind of microorganism, also comprises other bacterium under this kind
Strain, to make up the variation of bacterial strain under naturalness, this data base will be satisfied with the qualification requirement of microorganism relevant industries.Use this
Inventive method, associating multiple hospitals, scientific research institutions and each Culture Collection, collect microorganism fungus kind, and utilize mass spectrometric platforms, can
Building the microprotein fingerprint databases controlled oneself of Chinese, this spectrum library can be the Virulent Analysis, carefully of China's pathogenic microorganism
The research of the aspects such as the detection of bacterium typing, the molecular epidemiology of clone strain, bacterial resistance and resistance mechanism provides strong side
Help, be that China's biological and ecological methods to prevent plant disease, pests, and erosion establishes microbial identification basis simultaneously as national strategy resource.Use the inventive method, can be with structure
Build and comprise antibacterial, virus, fungus, actinomycetes, rickettsia, mycoplasma, chlamydia, spirillum etc. at least four thousand kinds, without ten thousand strains
Above bacterial strain standard diagram.
Accompanying drawing explanation
Fig. 1 is the analysis that the present invention builds storehouse collection of illustrative plates.
Fig. 2 is the characteristic protein peak information list that the present invention builds the MSP of storehouse collection of illustrative plates.
Fig. 3 is 30 the big enterobacteria collection of illustrative plates gathering and participating in building storehouse after relative analysis.
Fig. 4 is 30 the big enterobacteria collection of illustrative plates accumulation graphs building storehouse.
Fig. 5 is that 30 the big enterobacteria collection of illustrative plates stems gathered build one standard diagram of storehouse parameter synthesis.
Fig. 6 is that escherichia coli build the characteristic peak information list after storehouse completes.
Detailed description of the invention
The construction method bag in microbial polypeptide mass fingerprint storehouse based on flight time mass spectrum principle of the present invention
Include following detailed step:
The first step, the standardization of bacterial strain
1, the cultivation of bacterial strain
First the bacterial strain that will obtain from channels such as scientific research institutions, preservation centers, according to its physiological property, respectively at different culture media
On cultivate: such as fungus is on husky Borrow's agar plate, antibacterial on Colombia's blood plate or common blood plate 37 degree points
Not Pei Yang 48h and 24h, eisseria, Streptococcus etc. need inoculate chocolate agar plate, at the carbon dioxide environment of 5%
Middle cultivation 24h, obtains the bacterial strain of logarithmic (log) phase with this, and its principle is to strictly observe the condition of culture that various microorganism is set, carries
The qualification accuracy rate of high microorganism；
2, the preservation of bacterial strain
Owing to the database sharing time is longer, in order to ensure the survival that bacterial strain energy is steady in a long-term, need the bacterial strain that cultivation is obtained
Carry out preservation so that it is survival steady in a long-term；Conventional protective agent is: (1) acid compound: glutamic acid, aspartic acid, Herba Marsileae Quadrifoliae
Fruit acid etc.；(2) neutral compound: glucose, lactose, sucrose, Raffinose, sorbitol, inositol；(3) polymer substance and
Analyte: albumin, gelatin, peptone, algae；(4) natural mixture: skimmed milk, serum etc.；(5) other: ascorbic acid,
Azanol, glycerol etc..
Protectant criterion is selected to be: Strain survival is of long duration, variation probability rejuvenation low, easy.This builds data base's
Bacterial strain, protective agent uses the skimmed milk in natural mixture mostly, and its concentration is 2.5 ~ 10%, the bacterial strain such as part vibrio, mycoplasma
Preservation, on the basis of skimmed milk, with the addition of neutral compound and sodium chloride etc. as protective agent；Other special bacterial strain
Protective agent, according to bacterial strain physio-biochemical characteristics, screens in above protective agent.
3, the classification of bacterial strain and checking
1) the morphology checking of bacterial strain: first with morphological characteristics such as the size of bacterium colony, smooth degree, the most neat, the colors in edge
For foundation, it is determined that whether the bacterial strain of preservation is pure culture, secondly by microscope inspection, check the yin and yang attribute of Gram's staining, big
The microscopy characteristic of the bacterial strains such as little, form (for bacillus, coccus, diplococcus, vibrio, spore, spore), further ensures that the bacterium of preservation
Strain is single bacterial strain, i.e. ensures that the purity of preservation strain is 100%.
2) the taxonomy checking of bacterial strain:
In guaranteeing storehouse, error-free bacterium is the essential condition building data base, so before building data base, need to enter the bacterial strain of preservation
The multiple taxonomy method of row is identified, various methods compare mutually, it is ensured that build the correct of storehouse strain name；Applicant is to building storehouse
The verification method of bacterial strain uses: (1) form and biochemical identification: morphosis and biochemical character experiment, bio-chemical characteristics；(2)
Mass Spectrometric Identification: use the VITEK MS series of MALDI Biotyper, Mei Liai of Germany's Brooker.
3) molecules is identified: be respectively adopted DNA-DNA molecular hybridization, recA gene sequencing, GC% assay
Method, DNA marker technology based on 16S or 23S rRNA sequence, denaturing gradient gel electrophoretic analysis；Coding and automatization is used to reflect
Determine system (the more commonly used full-automatic biochemical assessing instrument VITEK 2 COMPACT, BD Phoenix-100 etc.) and fluorescence spectrum
Same bacterial strain is identified by method；When result is completely the same after identifying same bacterial strain according to different taxonomy, then carry out this
Storehouse is built in the collection of bacterial strain peptide mass fingerprinting spectrum, and the most do not carry out this bacterial strain builds storehouse.
Second step, builds the extraction of storehouse bacterial strain peptide fingerprinting spectrum
Owing to directly coating on target plate by bacterium colony, adding substrate HCCA(alpha-cyano, 4-hydroxycinnamic acid is as substrate, with 47.5%
Water, the trifluoroacetic acid of 2.5%, the mixed liquor of the acetonitrile of 50% is the HCCA saturated solution of solvent configuration), produced after drying
Crystallization speckle is uneven, and the peptide fingerprint protein graphical spectrum relative intensity, the position of m/z ratio that collect offset, collection of illustrative plates poor repeatability, so
Employing standard extraction method carries out building the extraction of storehouse peptide fingerprinting spectrum, and the equal non-metallic ion of equipment, big requiring to use when extracting
Molecularly Imprinted Polymers etc. are prone to protein bound centrifuge tube, the first-class experiment equipment of rifle and reagent, the peptide fingerprinting detected with guarantee
Compose true and reliable.
Under normal circumstances, can use following method that different strains carries out the extraction of peptide fingerprinting spectrum:
1, ethanol/formic acid method is suitable for the bacterium such as the antibacterial of the overwhelming majority, fungus, actinomycetes, mycoplasma, chlamydia, rickettsia
Strain.For shortening extraction time, improve the shell-broken effect of thalline, it is ensured that the accuracy of qualification result, in addition it is also necessary to carry out pre-to thalline
Processing, it specifically comprises the following steps that
A () (resistivity, close to 18.3M Ω/cm, adds generally for for building storehouse toward the ultra-pure water of addition certain volume in centrifuge tube
Enter 300ul ultra-pure water), add and cultivate the complete thalline building storehouse bacterial strain (for solid medium cultivation, a certain amount of list of picking
Bacterium colony, if liquid culture, is first centrifuged removing fluid medium, with ultra-pure water cyclic washing thalline to completely
Remove culture medium, add the resuspended thalline of ultra-pure water of 300ul), its turbidity scope control (or is simply chosen between 1 ~ 10 Maxwell
Take 5 ~ 10mg thalline in 300ul ultra-pure water), repeatedly with liquid-transfering gun piping and druming vortex, form bacteria suspension；
B () adds chromatographic grade dehydrated alcohol, add 900ul chromatographic grade ethanol when the present invention builds storehouse, be configured to 75% ethanol molten
Liquid, stands 2min after mixing, experiment proves that final concentration of alcohol, between 60% ~ 85%, stands more than one minute after vortex oscillation
(preferably 1 ~ 5 minute), it is possible to wash away the oil-soluble impurities on thalline；
When () present invention builds storehouse c, it is centrifuged 2min with 13000r/min, removes supernatant, after being again centrifuged, again remove supernatant, room temperature
Under be dried half an hour more than or use dry pot to carry out 35 ~ 40 DEG C of heating, experiment proves that 8000-15000r/min is centrifuged more than 2min
(at least twice), removes supernatant, stands till being completely dried, it is also possible to reaching requirement, its purpose predominantly removes ethanol, keeps away
Exempt to cause peptide fingerprinting spectral migration because being polymerized between ethanol and protein binding or albumen, cause the figure spectrum distortion of collection.
(d) add a certain amount of 40% ~ 90% formic acid (ultra-pure water is solvent, uses the first of 70% when the present invention builds collection of illustrative plates
Acid), dried thalline is completely covered, turbidity is not less than 1 Maxwell, after the resuspended mixing of thalline, static more than 2min, it is ensured that thalline
Breaking cellular wall fully (when the present invention builds storehouse, adds 20ul 70% aqueous formic acid, stands after repeatedly blowing and beating under liquid-transfering gun liquid level
2min).
E (present invention uses addition equal-volume acetonitrile to the acetonitrile of 0.8 ~ 1.5 times of formic acid volume of () addition, operation when building storehouse
For adding 20ul acetonitrile, under liquid-transfering gun liquid level, repeatedly blow and beat mixing), after mixing, static more than 2min, make albumen fully be dissolved into
In supernatant.
F () 10000-15000r/min is centrifuged more than 2min, take and put target after supernatant, and every 8-20 target position of strain bacterium point is each
Target site 0.5-2ul, dried some 0.5-2ul substrate, after being completely dried, upper machine is practiced shooting and (when the present invention builds storehouse, is used
13000r/min is centrifuged 2 minutes, takes 1ul supernatant o'clock on 96 hole target plates, and 10 sample spot of every strain bacterium point are after drying, each
Sample spot adds 1ul substrate, upper machine after being completely dried).
2,80%TFA extraction method
Although the sterilization effect of trifluoroacetic acid (TFA) is preferable, but its graph-spectrum quality gathered is slightly below ethanol/formic acid method, it is considered to
Identify, to build storehouse and later stage from now on, the biological safety used, with spore bacterial strain, rely on the bacterial strain of respiratory infectious with
And the bacterial strain that biological safety is more than two classes uses 80%TFA extraction method to extract peptide fingerprinting spectrum, its method particularly includes:
Take appropriate amount of sample (5 ~ 10mg), add 50ul 80%TFA, repeatedly blow and beat to the complete degeneration of sample, static 10 ~ 30min, add
Entering 100ul water and 150ul acetonitrile, after mixing, 13000r/min is centrifuged 2min, and absorption is checked target, adds 1ul substrate after being allowed to dry, complete
After white drying, upper machine is practiced shooting.
3rd step, builds the collection of storehouse bacterial strain peptide fingerprinting spectrum
1) the MALDI-TOF MS mass spectrometric platforms of determination employing applicant's unit independent research of collecting device (can certainly
Use the time-of-flight mass spectrometry of the VITEK MS series including the time-of-flight mass spectrometry of flex series of Brooker, Mei Liai) enter
The collection of row collection of illustrative plates, it is as follows that instrument adjusts parameter:
A () ionization laser source excites according to nitrogen, wavelength is 337nm；According to solid-state laser, wavelength is 355nm；
B the acquisition range of the peptide mass fingerprinting spectrum of () microbial standard spectrogram is between 2000-20000Da；
C the electric charge of () laser excitation is the polypeptide with a positive charge or albumen, use linear cation operator scheme, ion source
1 voltage 20 KV, ion source 2 voltage 17-20KV, electron lens voltage 5-10KV, between time delay extraction time 30 ~ 400ns
(ion source voltage, electron lens voltage, the parameter determination of time delay extraction time are entered with the resolution of peptide fingerprinting spectrum for foundation
Row sum-equal matrix, after parameter determination, resolution FWHM of the peptide fingerprinting spectrum at main body peak is more than 1000)；
D () laser frequency is that 40 ~ 100Hz(is usually 60Hz)；
E () detector voltage, between-2kv to-5kv, with substrate HCCA as background peaks during debugging, can determine detector pair
It reaches minimum voltage value during response；This magnitude of voltage can because of after detector life-time service decay and be gradually increased, therefore every
One week primary calibration to be carried out.
(f) instrument calibration: the laser number of blows gathering spectrogram is set as 40-200 time (optimal number of times is 100 times)；Often
Escherichia coli DH5a is used to carry out instrument calibration with the albumen of Myoglobin, the mixing of RNA (ribonucleic acid) enzyme before secondary data collection,
Choosing eight characteristic proteins being uniformly distributed in 2000-20000Da to calibrate instrument, the maximum error of its alignment is less than
150ppm, Calibration equation is:
y=ax2+ bx+c, wherein y represents m/z, x and represents the time；
2) each target spot of each bacterial strain of acquisition method gathers 3 ~ 5 collection of illustrative plates, and every collection of illustrative plates is by more than 4 times cumulative acquisitions, every time
Accumulated collection of illustrative plates to meet following condition: signal to noise ratio higher than 3.0, baseline is low and flat, main body peak intensity is at 2000-20000
Between, detector conversion time, the magnitude of voltage of 100mv be equivalent to 256 peak intensity (database build time, each target spot is usual
Adopting 3 spectrograms, every spectrogram adds up 6 times, and main body peak intensity is 6000-12000, and every strain bacterium gathers 30 spectrograms, is equivalent to this
30 spectrograms are obtained by 180 spectrograms are cumulative).
3) collection of illustrative plates processes and multiple collection of illustrative plates collected uses analysis software (can use the FlexAnalysis of Brooker
Analyze software) it is analyzed, first the characteristic peak of every collection of illustrative plates is compared, delete being clearly distinguishable from statistical significance
The collection of illustrative plates of other collection of illustrative plates, and delete underproof collection of illustrative plates, qualified collection of illustrative plates is retained, and collection of illustrative plates number is more than 20, is unsatisfactory for again carrying
Take collection, to reach the requirement of Database.As it is shown in figure 1, need the collection of illustrative plates indicated that deletes arrow, at the collection of illustrative plates collected
In, only these two are clearly distinguishable from other collection of illustrative plates, it should be that sampling error causes；
4th step, builds storehouse
1) the bacterial strain collection of illustrative plates of the 3rd step collection is carried out parameter determination
Standard diagram parameter is provided that
A) maximum error at single TuPu method peak is that 1500 ~ 2400ppm(is usually arranged as 200 ppm)；Due to mass spectrometer
Weighing peptide fingerprinting time spectrum, itself there is deviation, such as true peptide quality is 3000Da, should through its molecular mass of mass-spectrometer measurement
Between 2998Da to 3002Da, therefore should judge whether peptide quality to be determined indicates same albumen by this parameter,
Such as (2998-3000)/3000=667, less than 2000ppm, therefore the characteristic peak of 2998Da is owing to the error of instrument causes
Difference between measured value and actual value.
B) error of standard peptide quality fingerprinting reference spectrum is created at 200-300ppm；The structure of standard peptide mass fingerprint
Build, be that more than at least 20 qualified collection of illustrative plates synthesize a standard diagram (being called for short MSP) by after FlexAnalysis analyzes.
As a example by the characteristic peak of 5954Da in Fig. 1, judge that the characteristic peak fluctuated within the specific limits is instruction same egg through the first step (a)
In vain, if judging this feature peak measured value instruction same characteristic protein of 28 collection of illustrative plates through (a), by these 28 collection of illustrative plates
The practical measurement quality of the characteristic peak of 5954Da is averaged, then (measured value-meansigma methods)/meansigma methods ＜ of every collection of illustrative plates
(200-300ppm), then it is assumed that this collection of illustrative plates meets requirement for construction data base, participate in structure MSP, otherwise be then not involved in the structure of MSP,
Now need to recalculate the meansigma methods of this 5954Da characteristic peak, repeating previous step and calculate, until terminating, finally meeting this feature
The meansigma methods of the actual measurement albumen of the collection of illustrative plates of parameter, is this polypeptide or the albumen m/z value in the MSP built.
C) minimal graph spectral frequency: 25%；Still as a example by the characteristic peak of 5954Da in Fig. 1, after (a) and (b) two step judges, if
The collection of illustrative plates of 30 opening and closing lattice only has 20 structures participating in MSP, then the spectrogram frequency of occurrences at this feature peak is: 20/30=66.7%,
More than 25%, meet parameter request, the most undesirable, give up this feature peak.
(d) spectrogram maximum peak number: 70-80；The standard spectrum of every warehouse-in minimum by more than 20 collection of illustrative plates through normalization,
Smooth, base wavelet, then after (a) and (b), (c) three restriction choice of parameters, by characteristic peak intensity, error size, noise
The isoparametric comprehensive grading of ratio, is ranked up from high to low, picks out 70-80 characteristic peak and forms the MSP of this bacterial strain.Wherein count
Purpose selects can be in conjunction with the collection collection of illustrative plates of display in FlexAnalysis, and whether characteristic peak number completely includes
The characteristic peak of FlexAnalysis display collection of illustrative plates, selects according to actual characteristic peak number mesh.
2) forming a form after (a) and (b), (c), (d) four step screenings determine, it is contained within average matter lotus
Ratio, characteristic peak intensity, characteristic peak weight, the characteristic peak frequency of occurrences, participate in building a series of common feature peaks such as collection of illustrative plates quantity in storehouse,
As shown in the table of figure 2.
The standard diagram determined is unified with strain name, and marks out the banking process of this bacterial strain in detail, complete
This bacterial strain build storehouse；Other bacterial strain banking process the like, until completing the structure in microbial polypeptide mass fingerprint storehouse.
Below as a example by escherichia coli, describe the banking process of this bacterial strain in detail:
1, the cultivation of storehouse bacterial strain is built: escherichia coli are inoculated on blood plate cultivation, cultivate 24h for 37 degree；
2, the extraction of storehouse bacterial strain is built: in (1), past centrifuge tube, add the ultra-pure water of 300ul；(2) 3 ~ 5 escherichia coli list bacterium of picking
Falling, its turbidity scope control is in 5 Maxwells, and abundant vortex forms bacteria suspension；(3) the chromatographic grade dehydrated alcohol of 900ul, vortex are added
2 minutes are stood after vibration；(4) 13000r/min is centrifuged 2min, removes supernatant, is repeated once centrifugal, stands more than 20min under room temperature
To being completely dried.(5), after adding the resuspended mixing of formic acid thalline of 70% of 20ul, 2min is stood.(6) 20ul acetonitrile is added, mixing
After, stand 2min.(7) 13000r/min is centrifuged 2min, puts target after taking supernatant, puts 10 target position, each target site 1ul, is dried
Rear 1ul substrate, after being completely dried, upper machine is practiced shooting；
3, the confirmation of instrument parameter: using nitrogen excitation source, wavelength is 377nm, and the quality acquisition range of microbial identification is
Between 2000-20000Da, linear cation operator scheme, ion source 1 voltage 20 KV, ion source 2 voltage 18.1KV, electronics is saturating
Mirror voltage 6KV, time delay extraction time is 150ns；Laser frequency is 60HZ, and detector voltage, at negative 2673V, gathers swashing of spectrogram
Light number of blows is set to 100.Escherichia coli DH5a and Myoglobin, the egg mix of RNA (ribonucleic acid) enzyme is used before data acquisition
In vain instrument is calibrated；
4, building the collection of storehouse collection of illustrative plates and build storehouse: each target spot gathers 3 collection of illustrative plates, every collection of illustrative plates cumulative is obtained by 6 times, participates in every time
Cumulative collection of illustrative plates meets: signal to noise ratio higher than 3.0, baseline is low and flat, main body peak intensity is between 6000-12000.30 collected
Open collection of illustrative plates, to analyzing, software FlexAnalysis deletes the spectrogram being clearly distinguishable from other collection of illustrative plates on statistical significance, close
Lattice spectrogram carries out building storehouse, as shown in Figure 3,4.
Build standard peptide quality references collection of illustrative plates parameter and be provided that maximum error 2000ppm at single TuPu method peak；
Create the error of standard peptide quality references collection of illustrative plates at 200ppm；Minimal graph spectral frequency 25%；Collection of illustrative plates maximum peak number 70；Warehouse-in ginseng
Examine spectrum and formed containing average mass-to-charge ratio, characteristic peak strong after normalization, smooth, base wavelet, characteristic peak are selected by 30 spectrograms
Degree, characteristic peak weight, the characteristic peak frequency of occurrences, participate in building the list at a series of common feature peaks such as spectrogram quantity in storehouse, such as Fig. 5
Shown in Fig. 6 table.
Claims (2)
1. the construction method in a microbial polypeptide mass fingerprint storehouse based on flight time mass spectrum principle, it is characterised in that bag
Include following step:
The first step, the standardization of bacterial strain
First the bacterial strain of acquisition is carried out pure training according to respective physiological habit respectively under specific culture medium and condition of culture
Support, obtain the bacterial strain of logarithmic (log) phase successively；And bacterial strain cultivation obtained uses protective agent to carry out preservation so that it is steady in a long-term deposits
Live；
The bacterial strain survived preservation by morphology is verified, it is ensured that the purity of bacterial strain is 100%；By taxonomy to preservation
The bacterial strain of survival is verified, it is ensured that the title of bacterial strain is correct；By distinct methods, same bacterial strain is carried out molecules mirror
Fixed, it is ensured that the concordance of its qualification result；
Second step, builds the extraction of storehouse bacterial strain peptide fingerprinting spectrum
The first step confirms to meet the antibacterial of conditions for building groundwater reservoir, fungus, actinomycetes, mycoplasma, chlamydia, rickettsia bacterial strain enter
Row pretreatment, inactivates thalline and Lipid dissolution, uses ethanol/first acidity extraction peptide fingerprinting spectrum after drying；Will be with
The bacterial strain of spore, the bacterial strain relying on respiratory infectious and biological safety bacterial strain more than two classes use 80%TFA extraction method
Extraction peptide fingerprinting is composed；
3rd step, builds the collection of storehouse bacterial strain peptide fingerprinting spectrum
1) determination of collecting device uses MALDI-TOF MS mass spectrometric platforms to carry out the collection of collection of illustrative plates, and it is as follows that instrument adjusts parameter:
A () ionization laser source excites according to nitrogen, wavelength is 337nm；According to solid-state laser, wavelength is 355nm；
B the acquisition range of the peptide mass fingerprinting spectrum of () microbial standard spectrogram is between 2000-20000Da；
C the electric charge of () laser excitation is the polypeptide with a positive charge or albumen, use linear cation operator scheme, ion source
1 voltage 20 KV, ion source 2 voltage 17-20KV, electron lens voltage 5-10KV, time delay extraction time be 30-400ns it
Between；
D () laser frequency is 40-100HZ；
E () detector voltage, between-2kv to-5kv, with substrate HCCA as background peaks during debugging, determines detector and reaches it
To minimum voltage value during response；
(f) instrument calibration: the laser number of blows gathering spectrogram is set as 40-200 time；Use large intestine bar before data acquisition every time
Bacterium DH5a carries out instrument calibration with the albumen of Myoglobin, the mixing of RNA (ribonucleic acid) enzyme, chooses eight and is uniformly distributed in
Instrument is calibrated by the characteristic protein of 2000-20000Da, and the maximum error of its alignment is less than 150ppm, and Calibration equation is:
y=ax2+ bx+c, wherein y represents m/z, x and represents the time；
2) each target spot gathering each bacterial strain gathers 3 ~ 5 collection of illustrative plates, and every collection of illustrative plates, by more than 4 times cumulative acquisitions, participates in every time
Cumulative collection of illustrative plates to meet following condition: signal to noise ratio higher than 3.0, baseline is low and flat, main body peak intensity between 2000-20000,
The satisfactory spectrogram of each bacterial strain is at 20 ~ 40；
3) collection of illustrative plates processes and is analyzed in analyzing software by multiple collection of illustrative plates collected, the first characteristic peak to every collection of illustrative plates
Compare, delete the collection of illustrative plates being clearly distinguishable from other collection of illustrative plates on statistical significance, and delete underproof collection of illustrative plates, qualified figure
Spectrum is retained, and collection of illustrative plates number is more than 20, is unsatisfactory for need to again extracting collection, to reach requirement for construction data base；
4th step, builds storehouse
1) the bacterial strain collection of illustrative plates of the 3rd step collection is carried out parameter determination
Standard diagram parameter is provided that maximum error 1500-2400ppm at a) single TuPu method peak;B () creates standard peptide
The error of quality fingerprinting reference spectrum is at 200-300ppm；(c) minimum spectrogram frequency: 25%；(d) spectrogram maximum peak number: 70-
80；
2) the standard spectrogram determined is unified with strain name, and mark the banking process of this bacterial strain, complete this bacterial strain
Build storehouse；Other bacterial strain banking process the like, complete the structure in microbial polypeptide mass fingerprint storehouse.
The structure side in microbial polypeptide mass fingerprint storehouse based on flight time mass spectrum principle the most according to claim 1
Method, it is characterised in that: described second step to build storehouse bacterial strain peptide fingerprinting spectrum extract time, antibacterial, fungus, actinomycetes, mycoplasma, clothing
Substance, the preprocess method of rickettsia bacterial strain be:
The first step, the thalline that picking is appropriate from culture medium, use ultra-pure water to suspend, mix；
Second step, adds dehydrated alcohol, fully mixes, and makes the volume fraction of dehydrated alcohol in mixed liquor reach 70 ~ 80%, stands 1
~ 5 minutes；
3rd step, the mixed solution 10000 ~ 15000rpm/min rotating speed after being stood by second step is centrifuged 3 ~ 5 minutes, goes
Clearly；
4th step, by thalline constant temperature incubation 5 ~ 25 minutes under the conditions of 40 DEG C ± 1 DEG C of precipitation.
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