Matrix Assisted Desorption/Ionization Mass Spectrometry MALDI-MS Matrix Assisted Desorption/Ionization Mass Spectrometry Phillip Mnirajd

Introduction Mass Spectrometry (MS) Limitations New technique Vital tool used to characterize and analyze molecules Limitations Biomolecules and organic macromolecules are fragile Molecular ions or meaningful fragments were limited to only 5- 10 kDa at the time New technique In 1987, Michael Karas and Franz Hillenkamp successfully demonstrated the use of a matrix to ionize high molecular weight compounds [1].

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 matrix The presence of an aromatic matrix causes the large molecules to ionize instead of decomposing.

MALDI The mechanism remains uncertain It may involve absorption of light by the matrix Transfer of this energy to the analyte which 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 [2].

MALDI The 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.

MALDI see reference 3

MALDI Matrix The 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 [4].

MALDI Matrix According to Sigma Aldrich, the matrix must meet the following properties and requirements [5]: Be able to embed and isolate analytes (e.g. by co-crystallization) Be soluble in solvents compatible with analyte Be vacuum stable Absorb the laser wavelength Cause co-desorption of the analyte upon laser irradiation Promote analyte ionization

MALDI Matrix Reference 5

MALDI Matrix Reference 6 http://www.hopkinsmedicine.org/mams/mams/middleframe_files/teaching_files/me330.884/2005/ms2005-lecture-2-basic-maldi-esi.pdf Reference 6

MALDI Matrix For 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 30- 55 kDa [4].

MALDI Laser Numerous gas and solid state lasers have been developed for use in MALDI. Most MALDI devices use a pulsed UV laser N2 source at 337 nm neodymium-yttrium aluminum garnet (Nd:YAG) emits at 355 nm and gives a longer pulse time IR lasers are also used The most common IR laser is the erbium doped-yttrium aluminum garnet (Er:YAG) Emits at 2.94 micrometer it is “softer” than the UV, which is useful for certain biomolecules matrices available for IR absorption are limited

MALDI Laser Reference 5

MALDI Laser The MALDI method uses a pulse laser Laser fires in intervals Pulsed laser produces individual group of ions 1st pulse=1st group of ions 2nd pulse= 2nd group of ions, etc. Each group of ions generated are detected With continuous pulsing, the signal resolution increases

Time Mass Detectors The 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 [7] 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 http://www.kore.co.uk/mtof.htm

Reflectron Design in TOF-MS The TOF detector is also equipped with a reflectron, or an ion mirror The reflectron deflects the ion using an electric field and increases the path length, improving signal resolution [7]. Figure from: Muddiman, D. C.; Bakhtiar, R.; Hofstadler, S. A. J. Chem. Educ. 1997, 74, 1289.

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 hyperbolic A narrow range of m/z’s have stable trajectories through the quadrupole Ion motions governed by set of Mathieu equations Scanning the quadrupole generates the mass spectrum see reference 8

Quadrupole Mass Filter (QMF)

TOF vs QMF TOF and QMF are both used in MALDI QMF detectors are used more in teaching application Cheaper than TOF High accuracy and resolution not imperative TOF is the most typical detector used in research High mass limit

MALDI Advantages Gentle Ionization technique High molecular weight analyte can be ionized Molecule need not be volatile Sub-picomole sensitivity easy to obtain Wide array of matrices see reference 8

MALDI Disadvantages MALDI matrix cluster ions obscure low m/z species (<600) Analyte must have very low vapor pressure Pulsed nature of source limits compatibility with many mass analyzers Coupling MALDI with chromatography can be difficult Analytes that absorb the laser can be problematic Fluorescein-labeled peptides see reference 8

TOF Advantages All ions detected at once High mass accuracy and resolving power possible Reasonable performance for cost <5 ppm mass accuracy and >20,000 resolving power commercially available High mass, low charge ions not a problem Theoretically unlimited mass range Reference 8

TOF Disadvantages High vacuum required for resolution and accuracy (<10-7 torr) Complex vacuum system necessary Must be recalibrated often Temperature and voltage fluctuations alter flight times Fast detectors prone to saturation Long flight tubes for high resolving power can make instruments large Reference 8

QMF Advantages Very simple to implement Low cost (<$100k) Moderate vacuum required (~10-5 torr) Small size Most common MS in use Reference 8

QMF Disadvantages Limited mass range (up to m/z 4,000) Limited resolving power and mass accuracy Scanning limits sensitivity and speed Quad can rapidly jump between select m/z ratios for increased speed & sensitivity Refrence 8

Applications of MALDI Applications of MALDI mass spectrometry [9] Peptides and proteins Synthetic polymers Oligonucleotides Oligosaccharides Lipids Inorganics Bacterial identification Used especially Proteomics

Synthetic Polymer Analysis Using MALDI-TOF-MS MS spectrum of polybutylene adipate [7] In trans-3-indoleacrylic acid matrix Oligomer distribution is resolved Avg mol mass=4525 Da All ions are singly charged Distance between oligomers is mass of the repeating unit

Bacterial Identification Rapid bacterial identification is useful in diagnosing disease, monitoring contamination, etc. Important to identify related species Also identify strains in complex matrices Identified by: Biomarkers Cellular protein content MALDI-TOF-MS

Bacterial Identification MALDI-TOF-MS uses crude protein extract requiring minimal sample preparation Masses obtained of unknown is compared to experimentally determined signals Ions are specific to genus, species, or strain of bacteria MALDI-TOF-MS can determine mass of proteins of 1-40 kDa [10] Accuracy of  0.1% Due to the variability in percent composition of the isotopes

Bacterial Identification US Patent #6177266 B1 [10] January 23, 2001 United States of America as represented by the Secretary of the Army “Rapid Identification of Bacteria By Mass Spectrometry” Provides method to identify bacteria Genus, species, strain Bacteria identification on whole cells Provide library of biomarkers

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.

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 lyophilized Matrix 10 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).

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 cells Small quantity (0.1-0.2 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.

Bacterial Identification Mass spectral analysis of protein extracts Distinguishes among 4 strains of Bacillus

Bacterial Identification Mass spectral data of whole, intact cells Capable of detecting virulent and non virulent strains Bacillus REV-1 and Abortus

Bacterial Identification Comparison of two tables show common biomarkers and unique biomarkers in Bacillus species Different 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

Bacterial Identification see reference 11

Conclusion MALDI-MS is a vital tool in mass analysis of biomolecules and organic macromolecules Detection limits of femtomole to attomole [7] Reproducibility is relative Complimentary technique to ESI (electrospray ionization)

References M. Karas, et al and F. Hillenkamp; International Journal of Mass Spectrometry and Ion Processes, 78; 1987, p53. “Matrix Assisted Laser Desorption Ionization (MALDI).” http://www.tau.ac.il/lifesci/units/proteomics/voyager.html (6/18/2009). “MALDI-TOF Mass Analysis.” http://www.protein.iastate.edu/maldi.html (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.” http://www.sigmaaldrich.com/img/assets/4242/fl_analytix6_2001_new.pdf (6/17/09). “Lecture 2: Basic Maldi and Electrospray Theory.” http://www.hopkinsmedicine.org/mams/mams/middleframe_files/teaching_files/me330.884/2005/ms20 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.” http://www.sigmaaldrich.com/analytical-chromatography/spectroscopy/maldi-mass.html (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”; http://www.mcponline.org/cgi/reprint/7/2/448 (6/19/09)