Matrix-assisted laser desorption/ionization 1

Matrix-assisted laser desorption/ionization

MALDI TOF mass spectrometer

Matrix-assisted laser desorption/ionization (MALDI) is a softionization technique used in mass spectrometry, allowing theanalysis of biomolecules (biopolymers such as DNA, proteins,peptides and sugars) and large organic molecules (such aspolymers, dendrimers and other macromolecules), which tend tobe fragile and fragment when ionized by more conventionalionization methods. It is similar in character to electrosprayionization both in relative softness and the ions produced(although it causes many fewer multiply charged ions).

The MALDI is a two step process. First, desorption is triggered bya UV laser beam. Matrix material heavily absorbs UV laser light,leading to the ablation of upper layer (~micron) of the matrixmaterial. A hot plume produced during the ablation contains manyspecies: neutral and ionized matrix molecules, protonated anddeprotonated matrix molecules, matrix clusters and nanodroplets.The second step is ionization (more accurately protonation ordeprotonation). Protonation (deprotonation) of analyte moleculestakes place in the hot plume. Some of the ablated speciesparticipate in protonation (deprotonation) of analyte molecules.The mechanism of MALDI is still debated.

Matrix

UV MALDI Matrix List

Compound Other Names Solvent Wavelength(nm)

Applications

2,5-dihydroxy benzoic acid[1] DHB, Gentisicacid

acetonitrile, water,methanol, acetone,chloroform

337, 355, 266 peptides, nucleotides,oligonucleotides,oligosaccharides

3,5-dimethoxy-4-hydroxycinnamicacid[2][3]

sinapic acid;sinapinic acid;SA

acetonitrile, water,acetone, chloroform

337, 355, 266 peptides, proteins, lipids

4-hydroxy-3-methoxycinnamicacid[2][3]

ferulic acid acetonitrile, water,propanol

337, 355, 266 proteins

α-cyano-4-hydroxycinnamic acid[4] CHCA acetonitrile, water,ethanol, acetone

337, 355 peptides, lipids, nucleotides

Picolinic acid[5] PA Ethanol 266 oligonucleotides

3-hydroxy picolinic acid[6] HPA Ethanol 337, 355 oligonucleotides

The matrix consists of crystallized molecules, of which the three most commonly used are 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), α-cyano-4-hydroxycinnamic acid (alpha-cyano or alpha-matrix) and 2,5-dihydroxybenzoic acid (DHB). A solution of one of these molecules is made, often in a

Matrix-assisted laser desorption/ionization 2

mixture of highly purified water and an organic solvent (normally acetonitrile (ACN) or ethanol). Trifluoroaceticacid (TFA) may also be added. A good example of a matrix-solution would be 20 mg/mL sinapinic acid inACN:water:TFA (50:50:0.1).

Notation for cinnamic acid substitutions.

The identification of suitable matrix compounds isdetermined to some extent by trial and error, but theyare based on some specific molecular designconsiderations:•• They are of a fairly low molecular weight (to allow

facile vaporization), but are large enough (with alow enough vapor pressure) not to evaporate duringsample preparation or while standing in thespectrometer.

• They are often acidic, therefore act as a protonsource to encourage ionization of the analyte. Basic

matrices have also been reported.[7]

• They have a strong optical absorption in either the UV or IR range,[8] so that they rapidly and efficiently absorbthe laser irradiation. This efficiency is commonly associated with chemical structures incorporating severalconjugated double bonds, as seen in the structure of cinnamic acid.

•• They are functionalized with polar groups, allowing their use in aqueous solutions.•• They typically contain a chromophore.The matrix solution is mixed with the analyte (e.g. protein-sample). A mixture of water and organic solvent allowsboth hydrophobic and water-soluble (hydrophilic) molecules to dissolve into the solution. This solution is spottedonto a MALDI plate (usually a metal plate designed for this purpose). The solvents vaporize, leaving only therecrystallized matrix, but now with analyte molecules embedded into MALDI crystals. The matrix and the analyteare said to be co-crystallized. Co-crystallization is a key issue in selecting proper matrix to obtain good quality massspectrum of analyte of interest.

LaserMALDI techniques typically employ the use of UV lasers such as nitrogen lasers (337 nm) and frequency-tripled andquadrupled Nd:YAG lasers (355 nm and 266 nm respectively). Although not as common, infrared lasers are useddue to their softer mode of ionization. IR-MALDI also has the advantage of greater material removal (useful forbiological samples), less low-mass interferences, and compatibility with other matrix-free laser desorption massspectrometry methods.[9]

Ionization mechanismThe laser is fired at the matrix crystals in the dried-droplet spot. The matrix absorbs the laser energy and it is thoughtthat primarily the matrix is desorbed and ionized (by addition of a proton) by this event. The matrix is then thoughtto transfer proton to the analyte molecules (e.g., protein molecules), thus charging the analyte.[10] Ions observed afterthis process consist of a neutral molecule [M] and an added or removed ion. Together, they form a quasimolecularion, for example [M+H]+ in the case of an added proton, [M+Na]+ in the case of an added sodium ion, or [M-H]- inthe case of a removed proton. MALDI is capable of creating singly charged ions, but multiply charged ions([M+nH]n+) can also be created, as a function of the matrix, the laser intensity and/or the voltage used. Note thatthese are all even-electron species. Ion signals of radical cations (photoionized molecules) can be observed, e.g., incase of matrix molecules and other organic molecules.

Matrix-assisted laser desorption/ionization 3

Atmospheric pressure matrix-assisted laser desorption/ionizationAtmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) is an ionization technique (ionsource) that in contrast to vacuum MALDI operates at normal atmospheric environment.[11] The main differencebetween vacuum MALDI and AP-MALDI is the pressure in which the ions are created. In vacuum MALDI, ions aretypically produced at 10 mTorr or less while in AP-MALDI ions are formed in atmospheric pressure. In the past themain disadvantage of AP MALDI technique compared to the conventional vacuum MALDI has been its limitedsensitivity. With the introduction of AP MALDI Pulsed Dynamic Focusing (PDF) by MassTech, Inc. (Columbia,MD, USA) in 2004, ions are transferred into the mass spectrometer with high efficiency and attomole quantities ofpeptides has been reported in the peer-reviewed literature.[12]

AP-MALDI is used in mass spectrometry (MS) in a variety of applications ranging from proteomics to drugdiscovery. Popular topics that are addressed by AP-MALDI mass spectrometry include: proteomics; mass analysis ofDNA, RNA, PNA, lipids, oligosaccharides, phosphopeptides, bacteria, small molecules and synthetic polymers,similar applications as available also for vacuum MALDI instruments.The AP-MALDI ion source is easily coupled to an ion trap mass spectrometer[13] or any other MS system equippedwith ESI (electrospray ionization) or nanoESI source.

Mass spectrometer

Sample target for a MALDI mass spectrometer

The type of a mass spectrometer mostwidely used with MALDI is the TOF(time-of-flight mass spectrometer), mainlydue to its large mass range. The TOFmeasurement procedure is also ideallysuited to the MALDI ionization processsince the pulsed laser takes individual 'shots'rather than working in continuous operation.MALDI-TOF instrument or reflectron isequipped with an "ion mirror" that reflectsions using an electric field, thereby doublingthe ion flight path and increasing theresolution. Today, commercial reflectronTOF instruments reach a resolving powerm/Δm of well above 20'000 FWHM(full-width half-maximum, Δm defined asthe peak width at 50% of peak height).

MALDI has been coupled with IMS-TOF MS to identify phosphorylated and non-phosphorylated peptides.[14]

MALDI-FT-ICR MS has been demonstrated to be a useful technique where high resolution MALDI-MSmeasurements are desired.[15]

HistoryThe term matrix-assisted laser desorption ionization (MALDI) was coined in 1985 by Franz Hillenkamp, Michael Karas and their colleagues.[16] These researchers found that the amino acid alanine could be ionized more easily if it was mixed with the amino acid tryptophan and irradiated with a pulsed 266 nm laser. The tryptophan was absorbing the laser energy and helping to ionize the non-absorbing alanine. Peptides up to the 2843 Da peptide melittin could be ionized when mixed with this kind of “matrix”.[17] The breakthrough for large molecule laser desorption

Matrix-assisted laser desorption/ionization 4

ionization came in 1987 when Koichi Tanaka of Shimadzu Corp. and his co-workers used what they called the “ultrafine metal plus liquid matrix method” that combined 30 nm cobalt particles in glycerol with a 337 nm nitrogen laserfor ionization.[18] Using this laser and matrix combination, Tanaka was able to ionize biomolecules as large as the34,472 Da protein carboxypeptidase-A. Tanaka received one-quarter of the 2002 Nobel Prize in Chemistry fordemonstrating that, with the proper combination of laser wavelength and matrix, a protein can be ionized.[19] Karasand Hillenkamp were subsequently able to ionize the 67 kDa protein albumin using a nicotinic acid matrix and a266 nm laser.[20] Further improvements were realized through the use of a 355 nm laser and the cinnamic acidderivatives ferulic acid, caffeic acid and sinapinic acid as the matrix.[2] The availability of small and relativelyinexpensive nitrogen lasers operating at 337 nm wavelength and the first commercial instruments introduced in theearly 1990s brought MALDI to an increasing number of researchers.[21] Today, mostly organic matrices are used forMALDI mass spectrometry.

Applications

BiochemistryIn proteomics, MALDI is used for the rapid identification of proteins isolated by using gel electrophoresis:SDS-PAGE, size exclusion chromatography, affinity chromatography, strong/weak ion exchange, isotope codedprotein labelling (ICPL),and two-dimensional gel electrophoresis. Peptide mass fingerprinting is the most popularanalytical application of MALDI-TOF mass spectrometers. MALDI TOF/TOF mass spectrometers are used to revealamino acid sequence of peptides using post-source decay or high energy collision-induced dissociation (further usesee mass spectrometry).Loss of sialic acid has been identified in papers when DHB has been used as a matrix for MALDI MS analysis ofglycosylated peptides. Using sinapinic acid, 4-HCCA and DHB as matrices, S. Martin studied loss of sialic acid inglycosylated peptides by metastable decay in MALDI/TOF in linear mode and reflector mode.[22] A group atSHIMADZU CORPORATION derivatized the sialic acid by an amidation reaction as a way to improve detectionsensitivity [23] and also demonstrated that ionic liquid matrix reduces a loss of sialic acid during MALDI/TOF MSanalysis of sialylated oligosaccharides.[24] THAP,[25] DHAP,[26] and a mixture of 2-aza-2-thiothymine andphenylhydrazine [27] have been identified as matrices that could be used to minimize loss of sialic acid duringMALDI MS analysis of glycosylated peptides.It has been reported that a reduction in loss of some post-translational modifications can be accomplished if IRMALDI is used instead of UV MALDI [28]

Organic chemistrySome synthetic macromolecules, such as catenanes and rotaxanes, dendrimers and hyperbranched polymers, andother assemblies, have molecular weights extending into the thousands or tens of thousands, where most ionizationtechniques have difficulty producing molecular ions. MALDI is a simple and fast analytical method that can allowchemists to rapidly analyze the results of such syntheses and verify their results.

Polymer chemistryIn polymer chemistry MALDI can be used to determine the molar mass distribution.[29] Polymers withpolydispersity greater than 1.2 are difficult to characterize with MALDI due to the signal intensity discriminationagainst higher mass oligomers.[30][31][32] A good matrix for polymers is dithranol and AgTFA. The sample must firstbe mixed with dithranol and the AgTFA added afterwards; otherwise the sample would precipitate out of solution.

Matrix-assisted laser desorption/ionization 5

MicrobiologyMALDI/TOF spectra are used for the identification of microorganisms such as bacteria or fungi. A colony of themicrobe in question is smeared directly on the sample target and overlayed with matrix. The mass spectra generatedare analyzed by dedicated software and compared with stored profiles. Species diagnosis by this procedure is muchfaster, more accurate and cheaper than other procedures based on immunological or biochemical tests. MALDI/TOFmay become the standard method for species identification in medical microbiological laboratories over the next fewyears.[33]

Reproducibility and performanceThe sample preparation for MALDI is important for both sensitivity, reproducibility, and quantification of massanalysis. Inorganic salts which are also part of protein extracts interfere with the ionization process. The salts can beremoved by solid phase extraction or by washing the dried-droplet MALDI spots with cold water. Both methods canalso remove other substances from the sample. The matrix-protein mixture is not homogenous because the polaritydifference leads to a separation of the two substances during co-crystallization. The spot diameter of the target ismuch larger than that of the laser, which makes it necessary to make many laser shots at different places of thetarget, to get the statistical average of the substance concentration within the target spot. The matrix chemicalcomposition, the addition of trifluoroacetic acid, formic acid, fructose, delay time between the end of laser pulse andstart of ion acceleration in the ion source (in vacuum MALDI sources), laser wavelength, UV energy (as well as itsdensity and homogeneity)in a focused light spot produced by pulsed laser, and the impact angle of the laser on thetarget are among critical parameters for the quality and reproducibility of the MALDI-TOF MS method.

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J. Mass Spectrom. Ion Processes 72 (111): 89–102. Bibcode 1991IJMSI.111...89S. doi:10.1016/0168-1176(91)85050-V.[2] Beavis RC, Chait BT (1989). "Matrix-assisted laser-desorption mass spectrometry using 355 nm radiation". Rapid Commun. Mass Spectrom.

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[7] (Fitzgerald et al., Anal. Chem. 65:3204-3211,1993)Fitzgerald, Michael C.; Parr, Gary R.; Smith, Lloyd M. (1993). "Basic matrixes for thematrix-assisted laser desorption/ionization mass spectrometry of proteins and oligonucleotides". Analytical Chemistry 65 (22): 3204–11.doi:10.1021/ac00070a007. PMID 8291672.

[8] Zenobi and Knochenmuss Mass Spectrometry Reviews 17: 337-366, 1998 Zenobi, Renato; Knochenmuss, Richard (1998). "Ion formation inMALDI mass spectrometry". Mass Spectrometry Reviews 17 (5): 337.doi:10.1002/(SICI)1098-2787(1998)17:5<337::AID-MAS2>3.0.CO;2-S.

[9] "Murray Group LSU" (http:/ / mass-spec. lsu. edu/ wiki/ index. php/ Infrared_MALDI). . Retrieved 2010-01-20.[10] Knochenmuss R (2006). "Ion formation mechanisms in UV-MALDI". Analyst 131 (9): 966–986. Bibcode 2006Ana...131..966K.

doi:10.1039/b605646f. PMID 17047796.[11] Laiko VV, Baldwin MA, Burlingame AL (2000). "Atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry".

Anal. Chem. 72 (4): 652–7. doi:10.1021/ac990998k. PMID 10701247.[12] Lee, Arnold. "Biological Applications of AP MALDI with Thermo Scientific Exactive Orbitrap MS" (http:/ / www. apmaldi. com/ images/

stories/ 005-111101-an30224-maldi-generalapp_6s_v4a_kp. pdf). 443-539-1710. Thermo Scientific. . Retrieved 17 June 2011. "In the past themain disadvantage of AP MALDI technique compared to the conventional vacuum MALDI has been its limited sensitivity. With theintroduction of AP MALDI Pulsed Dynamic Focusing (PDF) by MassTech, Inc. (Columbia, MD, USA) in 2004, ions are transferred into themass spectrometer with high efficiency and attomole quantities of peptides has been reported in the peer-reviewed literature"

Matrix-assisted laser desorption/ionization 6

[13] Laiko VV, Moyer SC, Cotter RJ (2000). "Atmospheric pressure MALDI/ion trap mass spectrometry". Anal. Chem. 72 (21): 5239–43.doi:10.1021/ac000530d. PMID 11080870.

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Spectrometry of Organic Molecules". Anal. Chem. 57 (14): 2935–9. doi:10.1021/ac00291a042.[17] Karas, M.; Bachman, D.; Bahr, U.; Hillenkamp, F. (1987). "Matrix-Assisted Ultraviolet Laser Desorption of Non-Volatile Compounds". Int

J Mass Spectrom Ion Proc 78: 53–68. doi:10.1016/0168-1176(87)87041-6.[18] Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. (1988). "Protein and Polymer Analyses up to m/z 100 000 by Laser

Ionization Time-of flight Mass Spectrometry". Rapid Commun Mass Spectrom 2 (20): 151–3. doi:10.1002/rcm.1290020802.[19] Markides, K; Gräslund, A. "Advanced information on the Nobel Prize in Chemistry 2002" (http:/ / nobelprize. org/ chemistry/ laureates/

2002/ chemadv02. pdf) (PDF). .[20] Karas M, Hillenkamp F (1988). "Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons". Anal. Chem. 60

(20): 2299–301. doi:10.1021/ac00171a028. PMID 3239801.[21] Karas, M.; Bahr, U. (1990). "Laser Desorption Ionization Mass Spectrometry of Large Biomolecules". Trends Anal. Chem. 9 (10): 321–5.

doi:10.1016/0165-9936(90)85065-F.[22][22] Huberty et al., Anal. Chem. 65:2791-2800, 1993[23][23] Sekiya et al., Anal. Chem. 77:4962-4968, 2005[24][24] Fukuyama et al., Anal. Chem. 80:2171-2179, 2008[25][25] Papac et al., Anal. Chem. 68:3215-3223, 1996[26][26] Harvey Mass Spectrometry Reviews 18: 349-451, 1999[27][27] Lattova et al., Rapid Communications in Mass Spectrometry 21: 1644-1650, 2007[28][28] Tajiri et al.,Anal. Chem. 81:6750-6755, 2009[29] W. Schrepp; Harald Pasch (2003). Maldi-Tof Mass Spectrometry of Synthetic Polymers (Springer Laboratory). Berlin: Springer-Verlag.

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[33] Seng P, Drancourt M, Gouriet F, La Scola B, Fournier PE, Rolain JM, Raoult D. (August 2009). "Ongoing revolution in bacteriology:routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry". Clin Infect Dis. 49 (4):552–3. doi:10.1086/600885. PMID 19583519.

Bibliography• Hillenkamp F, Karas M, Beavis RC, Chait BT (1991). "Matrix-assisted laser desorption/ionization mass

spectrometry of biopolymers". Anal. Chem. 63 (24): 1193A–1203A. doi:10.1021/ac00024a002. PMID 1789447.• Ragoussis J, Elvidge GP, Kaur K, Colella S (2006). "Matrix-Assisted Laser Desorption/Ionisation, Time-of-Flight

Mass Spectrometry in Genomics Research". PLoS Genet. 2 (7): e100. doi:10.1371/journal.pgen.0020100.PMC 1523240. PMID 16895448.

• Hardouin J (2007). "Protein sequence information by matrix-assisted laser desorption/ionization in-source decaymass spectrometry". Mass spectrometry reviews 26 (5): 672–82. doi:10.1002/mas.20142. PMID 17492750.

• Jasna Peter-Katalinic; Franz Hillenkamp (2007). MALDI MS: A Practical Guide to Instrumentation, Methods andApplications. Weinheim: Wiley-VCH. ISBN 3-527-31440-7. OCLC 180943017.

• W. Schrepp; Harald Pasch (2003). Maldi-Tof Mass Spectrometry of Synthetic Polymers (Springer Laboratory).Berlin: Springer-Verlag. ISBN 3-540-44259-6. OCLC 51330276.

Matrix-assisted laser desorption/ionization 7

External links• Primer on Matrix-Assisted Laser Desorption Ionization (MALDI) (http:/ / www. magnet. fsu. edu/ education/

tutorials/ tools/ ionization_maldi. html) National High Magnetic Field Laboratory

Article Sources and Contributors 8

Article Sources and ContributorsMatrix-assisted laser desorption/ionization  Source: http://en.wikipedia.org/w/index.php?oldid=499847477  Contributors: 28bytes, Alboyle, Andrei Stroe, Anradt, Aron Hennerdal, Bfigura,CWenger, Cacycle, Chris the speller, Clicketyclack, Coolguyche17, Ctdunstan, DoriSmith, Dragnmn, DragonflySixtyseven, EndersJ, Explicit, Fjpaffen, GLSmyth, Gene Nygaard, Grimlock,Guyler, H Padleckas, Hankwang, Headbomb, Hede2000, Ike9898, Ileresolu, Isfahani, J.delanoy, JOK, JonHarder, K Eliza Coyne, Kehrli, Kilo-Lima, Kkmurray, Kmurray, MarcoTolo, Martious,Michael Hardy, Nick Y., Nono64, ObfuscatePenguin, Oxymoron83, Pankratz23, Pleiade, Proteochem, Ramchandra1981, Rbeavis, Rjwilmsi, Salamurai, SaveTheWhales, Spondoolicks,StAnselm, Stone, Ternto333, TheBendster, Toftofmsms, Tregoweth, Vector Potential, Verak, WolfmanSF, 102 anonymous edits

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