Presentation on theme: "Matrix Assisted Desorption/Ionization Mass Spectrometry"— Presentation transcript:
1 Matrix Assisted Desorption/Ionization Mass Spectrometry MALDI-MSMatrix Assisted Desorption/Ionization Mass SpectrometryPhillip Mnirajd
2 Introduction Mass Spectrometry (MS) Limitations New technique Vital tool used to characterize and analyze moleculesLimitationsBiomolecules and organic macromolecules are fragileMolecular ions or meaningful fragments were limited to only kDa at the timeNew techniqueIn 1987, Michael Karas and Franz Hillenkamp successfully demonstrated the use of a matrix to ionize high molecular weight compounds .
3 MALDI Matrix Assisted Laser Desorption/Ionization (MALDI) Method where a laser is used to generate ions of high molecular weight samples, such as proteins and polymers.Analyte is embedded in to crystal matrixThe presence of an aromatic matrix causes the large molecules to ionize instead of decomposing.
4 MALDI The mechanism remains uncertain It may involve absorption of light by the matrixTransfer of this energy to the analytewhich then ionizes into the gas phase as a result of the relatively large amount of energy absorbed.To accelerate the resulting ions into a flight-tube in the mass spectrometer they are subjected to a high electrical field .
5 MALDIThe MALDI technique combined with a MS detector (MALDI-MS) became an indispensable tool in analysis of biomolecules and organic macromolecules.MALDI involves incorporation of the analyte into a matrix, absorption/desorption of laser radiation, and then ionization of the analyte.
7 MALDI MatrixThe analyte incorporation in to a suitable matrix is the first step of the MALDI process, and is an important feature of the MALDI method.A typical sample preparation involves using 10-6 M solution of the analyte mixed with 0.1 M solution of the matrix.The solvents are then evaporated in a vacuum of the MS, and the matrix crystallizes with the analyte incorporated .
8 MALDI MatrixAccording to Sigma Aldrich, the matrix must meet the following properties and requirements :Be able to embed and isolate analytes (e.g. by co-crystallization)Be soluble in solvents compatible with analyteBe vacuum stableAbsorb the laser wavelengthCause co-desorption of the analyte upon laser irradiationPromote analyte ionization
11 MALDI MatrixFor compounds that are not soluble in the standard solvents, a solventless method was developed, in particular for synthetic polymers.The method involves mixing the matrix and analyte powders that were ground in a mortar. The mixture is then applied to a MALDI target support and the spectrum is obtained. However, this particular method leads to increased fragmentation of ions and has a mass limit of kDa .
12 MALDI LaserNumerous gas and solid state lasers have been developed for use in MALDI.Most MALDI devices use a pulsed UV laserN2 source at 337 nmneodymium-yttrium aluminum garnet (Nd:YAG)emits at 355 nm and gives a longer pulse timeIR lasers are also usedThe most common IR laser is the erbium doped-yttrium aluminum garnet (Er:YAG)Emits at 2.94 micrometerit is “softer” than the UV, which is useful for certain biomoleculesmatrices available for IR absorption are limited
14 MALDI Laser The MALDI method uses a pulse laser Laser fires in intervalsPulsed laser produces individual group of ions1st pulse=1st group of ions2nd pulse= 2nd group of ions, etc.Each group of ions generated are detectedWith continuous pulsing, the signal resolution increases
15 Time Mass DetectorsThe typical detector used with MALDI is the time of flight mass detector (TOF-MS)TOF is a method where the ions are accelerated by an electric field, resulting in ions of the same strength to have the same kinetic energy The time it takes for each ion to traverse the flight tube and arrive at the detector is based on its mass-to-charge ratio; therefore the heavier ions have shorter arrival times compared to lighter ions
16 Reflectron Design in TOF-MS The TOF detector is also equipped with a reflectron, or an ion mirrorThe reflectron deflects the ion using an electric field and increases the path length, improving signal resolution .Figure from: Muddiman, D. C.; Bakhtiar, R.; Hofstadler, S. A. J. Chem. Educ , 74, 1289.
17 Quadrupole Mass Filter (QMF) QMF involves the generation of radio frequency (RF) and DC field between opposite pairs of 4 rods.Rods can be cylindrical or hyperbolicA narrow range of m/z’s have stable trajectories through the quadrupoleIon motions governed by set of Mathieu equationsScanning the quadrupole generates the mass spectrumsee reference 8
19 TOF vs QMF TOF and QMF are both used in MALDI QMF detectors are used more in teaching applicationCheaper than TOFHigh accuracy and resolution not imperativeTOF is the most typical detector used in researchHigh mass limit
20 MALDI Advantages Gentle Ionization technique High molecular weight analyte can be ionizedMolecule need not be volatileSub-picomole sensitivity easy to obtainWide array of matricessee reference 8
21 MALDI DisadvantagesMALDI matrix cluster ions obscure low m/z species (<600)Analyte must have very low vapor pressurePulsed nature of source limits compatibility with many mass analyzersCoupling MALDI with chromatography can be difficultAnalytes that absorb the laser can be problematicFluorescein-labeled peptidessee reference 8
22 TOF Advantages All ions detected at once High mass accuracy and resolving power possibleReasonable performance for cost<5 ppm mass accuracy and >20,000 resolving power commercially availableHigh mass, low charge ions not a problemTheoretically unlimited mass rangeReference 8
23 TOF DisadvantagesHigh vacuum required for resolution and accuracy (<10-7 torr)Complex vacuum system necessaryMust be recalibrated oftenTemperature and voltage fluctuations alter flight timesFast detectors prone to saturationLong flight tubes for high resolving power can make instruments largeReference 8
24 QMF Advantages Very simple to implement Low cost (<$100k) Moderate vacuum required (~10-5 torr)Small sizeMost common MS in useReference 8
25 QMF Disadvantages Limited mass range (up to m/z 4,000) Limited resolving power and mass accuracyScanning limits sensitivity and speedQuad can rapidly jump between select m/z ratios for increased speed & sensitivityRefrence 8
26 Applications of MALDI Applications of MALDI mass spectrometry  Peptides and proteinsSynthetic polymersOligonucleotidesOligosaccharidesLipidsInorganicsBacterial identificationUsed especiallyProteomics
27 Synthetic Polymer Analysis Using MALDI-TOF-MSMS spectrum of polybutylene adipate In trans-3-indoleacrylic acid matrixOligomer distribution is resolvedAvg mol mass=4525 DaAll ions are singly chargedDistance between oligomers is mass of the repeating unit
28 Bacterial Identification Rapid bacterial identification is useful in diagnosing disease, monitoring contamination, etc.Important to identify related speciesAlso identify strains in complex matricesIdentified by:BiomarkersCellular protein contentMALDI-TOF-MS
29 Bacterial Identification MALDI-TOF-MS uses crude protein extract requiring minimal sample preparationMasses obtained of unknown is compared to experimentally determined signalsIons are specific to genus, species, or strain of bacteriaMALDI-TOF-MS can determine mass of proteins of kDa Accuracy of 0.1%Due to the variability in percent composition of the isotopes
30 Bacterial Identification US Patent # B1 January 23, 2001United States of America as represented by the Secretary of the Army“Rapid Identification of Bacteria By Mass Spectrometry”Provides method to identify bacteriaGenus, species, strainBacteria identification on whole cellsProvide library of biomarkers
31 Bacterial Identification The present invention provides a method for generating unique mass spectral profiles for bacteria protein extracts or whole bacteria cells. These profiles contain proteinaceous biomarkers which distinguish between bacteria of different genera, species and strains. Comparable profiles are generated when the method is performed using different MALDI-TOF instruments from different manufactures.
32 Sample Preparation Bacteria Matrix supplied as γ-irradiated and lyophilized samples by the U.S. Army Laboratories at Dugway Proving Ground, Utah.Nonpathogenic bacteria cells of different strains were grown in- house by incubating for 24 hrs. at 37° C. on trypticase soy agar or nutrient agar plates, harvested and lyophilizedMatrix10 mg/ml of either 4-hydroxy-α-cyano-cinnamic acid (4 CHCA; 10 mg/ml) or 3,5-dimethoxy-4-hydroxy cinnamic acid (sinapinic acid) in an aqueous solvent solution comprising 0.1% aqueous trifluoroacetic acid (TFA) and acetronitrile in a ratio of 70/30 (v/v).
33 Sample Preparation Protein extracts For analysis of whole cells 1 μl of a protein extract was mixed with 9 μl of matrix solution.For analysis of whole cellsSmall quantity ( mg) of intact, whole cells are suspended were added to 20 μl of aqueous buffer, typically 0.1% trifluoroacetic acid, vortexed for 30 seconds, and 1 μl of the resulting suspension was either frozen for later use and thawed and combined with 9 μl of a matrix solution or used immediately.
34 Bacterial Identification Mass spectral analysis of protein extractsDistinguishes among 4 strains of Bacillus
35 Bacterial Identification Mass spectral data of whole, intact cellsCapable of detecting virulent and non virulent strainsBacillus REV-1 and Abortus
36 Bacterial Identification Comparison of two tables show common biomarkers and unique biomarkers in Bacillus speciesDifferent strains of a bacteria species can also be MALDI-TOF-MS analysis of protein and of mass spectral analysis of intact, whole cells by the above procedure also produced biomarkers which distinguished between bacteria at the genus, species and strain levels
38 ConclusionMALDI-MS is a vital tool in mass analysis of biomolecules and organic macromoleculesDetection limits of femtomole to attomole Reproducibility is relativeComplimentary technique to ESI (electrospray ionization)
39 ReferencesM. Karas, et al and F. Hillenkamp; International Journal of Mass Spectrometry and Ion Processes, 78; 1987, p53.“Matrix Assisted Laser Desorption Ionization (MALDI).” (6/18/2009).“MALDI-TOF Mass Analysis.” (6/18/2009).Jasna Peter-Katalinic; Franz Hillenkamp (2007). “MALDI MS: A Practical Guide to Instrumentation, Methods and Applications.” Weinheim: Wiley-VCH.“Maldi Mass Spectrometry.” (6/17/09).“Lecture 2: Basic Maldi and Electrospray Theory.” 05-lecture-2-basic-maldi-esi.pdf (6/20/2009).Muddiman, D. C.; Bakhtiar, R.; Hofstadler, S. A. J. Chem. Educ. 1997, 74, 1289.Karty, Johnathan A.” Introduction to Walk-Up Mass Spectrometry.” msf.chem.indiana.edu/.../Introduction%20to%20Mass%20Spectrometry%20july2008.ppt (6/21/09).“MALDI Mass.” (6/22/09).Krishnamurthy, T. U.S. Patent 6,177,266, 2001.Lee, Y. “Highly Efficient Classification and Identification of Human Pathogenic Bacteria By MALDI-TOF-MS”; (6/19/09)