2010-7-2-CAU-MALDI-TOF及其分类上的应用-2

2010/6/231WenFengCHEN (陈文峰)Application of MALDI TOF MS on the Taxonomy of Bacteria

Visiting Scholar of UHMAssociate Prof. of CAU

IntroductionPrincipleProcedure

Application

IntroductionIdentificationofbacteriabytraditionalmolecularandbiochemicalmethodsisrelativelyslowandtimeconsuming(and,inthecaseofmoleculartechniques,expensive).Methods for identification of bacteria and their shortcomings:Cultural methods (mostly requiring several days)IntroductionMorphologySelective and differential mediaChemotaxonomic methodsBiochemical tests

IntroductionSerological methods (mostly requiring one day)LatexagglutinationMethods for identification of bacteria and their shortcomings:Latex agglutinationELISA: Enzyme-linked immunosorbentassays IFAs: ImmunofluorescenceassaysImmunomagneticseparation IntroductionGenetic methods (mostly requiring one day)Nucleic acid hybridizationFluorescent in situ hybridization (FISH)

Methods for identification of bacteria and their shortcomings:

y()PCRRT-PCR, Multiplex PCR, Nested PCR,Arbitrarily primed (AP) PCRERIC, REP, BOX PCRRAPD, RFLP PCRReal-time PCRDNA microarrays2010/6/232IntroductionOther methods (requiring one to several days)Phage typingCapillary electrophoresisMethods for identification of bacteria and their shortcomings:pypFlow cytometryInfrared spectroscopy, including FTIRPAGE: Polyacrylamide gel electrophoresisARDRA: amplified ribosomal DNA restriction analysisRibotypingPFGE: Pulsed-field gel electrophoresisAFLP: amplified fragment length polymorphismIntroductionMethods for identification of bacteria and their shortcomings:

IntroductionAn alternative to these is chemotaxonomy–the identification of bacteria by protein markers. Mass spectrometry is a powerful tool for such identification. Principlep

MALDI：Matrix-assisted laser desorption/ionization

TOF：Time of FlightMS: Mass spectrometryAltWhat is MALDI TOF MS?Analyte: Biomolecules(proteins, peptides and sugars);Large organic molecules (polymers).

MatrixCrystallized molecules.3 most commonly used matrixes:SA: 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinicacid)HCCA:α-cyano-4-hydroxycinnamic acidDHB:2,5-dihydroxybenzoic acidPreparation:

Matrix+Highlypurified+Organicsolvent(acetonitrileor+ Highly purified H2O+ Organic solvent (acetonitrile or ethanol). Trifluoroaceticacid (TFA) may also be added. eg. 20 mg/mLSA in ACN:water:TFA(50:50:0.1).Organic solvent: to dissolve hydrophobic moleculesH2O: to dissolve hydrophilic moleculesMatrix: to protect the biomoleculefrom being destroyed by direct laser beam and to facilitate vaporization and ionization.2010/6/233MatrixAcidic, has polar groups (can be used in aqueous solutions).Proton source (to encourage ionization of the analyte)Strong optical absorption (UV or IR)SeveralconjugateddoublebondsSeveral conjugated double bonds

cinnamic acid(肉桂酸或苯丙烯酸)

nitrogen lasers (337 nm)LaserA nitrogen laseris a gas laser operating in the ultraviolet range (typically 337nm), using molecular nitrogen as its gain medium, pumped by an electrical discharge.

Principle of MALDI TOF MS Matrix crystalAcceleration areaSample inletIon detectorFlight pathLight ionsHigh ionsIonisation areaPrinciple of MALDI TOF MS The separation of charged particles of mass spectrometry is based on mass, charge, and flight path (time).

Vacuum chamberTime measurement

ProcedureCell culturing on solid medium TIMING ~6–48 h

(i) For cell culturing of Enterobacteriaceaesuch as E. coli on agar plates, incubate all dilutions for up to 2 days at an optimal temperature (generally between 28 and 37°C).

(ii) Resuspendbacteria from single colonies grown on the solid medium plate in 1 ml double distilled water.CRITICAL STEPFor good quality spectra, only one single colony should be applied. It is very important that the user always compares mass spectra of bacterial samples that were grown under similar conditions, particularly for bacterial analysis on the subspecies level. It is generally important that the bacteria used for comparative mass spectrometric analysis have CELL CULTUREgypppyentered the same growth phase (use samples that are either in the logor in the stationaryphase).

(iii) Centrifuge this sample in 1.5 ml tubes at 10,000g at room temperature for ~2 min and discard the supernatant.

(iv) The pellet can be additionally washed with 1 ml double distilled water; discard the supernatant again.However, in many cases, this step is not necessary. Continue with Step 2.! CAUTION Whatever culture conditions are applied, handle all biological samples as a potential source of pathogens. Use appropriate protective attire (lab coats, safety glasses, latex gloves and other conventional measures applied in the microbiology laboratory) and dispose all biohazardousmaterials properly.