Sample Preparation for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry
Sample Preparation for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry
Mass Spectrometry Letters. 2015. Jun, 6(2): 27-30
Copyright © 2015, Korean Society Mass Spectrometry
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  • Received : May 18, 2015
  • Accepted : June 11, 2015
  • Published : June 30, 2015
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Jeongkwon Kim

This article reviews the fundamentals of sample preparation used in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). MALDI is a soft ionization method used to generate analyte ions in their intact forms, which are then detected in MS analysis. MALDI-MS boasts fast analysis times and easy-to-use operation. The disadvantages of MALDI-MS include the occurrence of matrix-associated peaks and inhomogeneous distribution of analyte within the matrix. To overcome the disadvantages of MALDI-MS, various efforts have been directed such as using different matrices, novel matrix systems, various additives, and different sample preparation methods. These various efforts will be discussed in detail. This article will benefit those who would like to obtain basic knowledge of MALDI sample preparation and those who would like to use MALDI-MS in their chemical analyses.
Matrix-assisted laser desorption/ionization (MALDI) is an ionization process that is capable of ionizing nonvolatile, high-molecular-weight compounds in their intact form for subsequent mass spectrometry (MS) analysis. MALDI-MS is a relatively easy method for determining the molecular weight of a compound. Due to its ease of operation, fast analysis times, high mass accuracy, and high mass resolution, MALDI-MS is commonly used for rapid analyses of proteins, peptides, synthetic polymers, glycans, and other macromolecules. In the MALDI process, a laser beam impinges a spot on a stainless steel plate where the sample has been dispersed in a large excess of matrix material. The sample is ionized by interactions with the excited matrix, is detached from the spot, and detected as an ion. Detailed ionization mechanisms are described in other review articles. 1 , 2 Time-of-flight (TOF) analyzers are currently the most commonly used to analyze these ions generated from MALDI, where the time required for an ion to move from the ionization region to a detector region is used to determine its mass-to-charge ( m/z ) ratio. Currently, the most commonly used laser sources are nitrogen lasers at 337 nm and Nd:YAG lasers at 355 nm. The typical spatial resolution of a commercial MALDI TOF instrument, as limited by the diameter of the impinging laser beam on the sample surface, is around 20 µm. 3 Special settings can be applied to attain spatial resolutions down to 5 µm. 3 The typical repetition rate of a MALDI laser source is 1-kHz. 4 Figure 1 shows a schematic diagram of the MALDI process.
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Schematic diagram of the MALDI process.
The phase “matrix-assisted laser desorption” was first used publically in 1985 by Michael Karas, Doris Bachmann, and Franz Hillenkamp to describe the mass spectrum of alanine from a 1:1 mixture of alanine and tryptophan using a desorption laser at 266 nm. 5 Tryptophan was regarded as an absorbing matrix that resulted in the ionization of nonabsorbing alanine. The “ultrafine metal plus liquid matrix method”, by Koichi Tanaka, 6 used a 337-nm nitrogen laser to ionize large molecules such as proteins and synthetic polymers. His contribution to the first large-molecule analysis using laser desorption ionization won him the 2002 Nobel Prize in Chemistry, shared with John Bennett Fenn and Kurt Wüthrich.
This article overviews the sample preparation in MALDI-MS, covering common matrices, novel matrix systems, unique additives and different sample preparation methods.
Common matrices used in MALDI
An optimum matrix is determined by the properties of the analyte. Currently, 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), and sinapic acid (SA) are the most commonly used matrix materials. Recently, many other organic materials have been evaluated, including 2,4,6-trihydroxyacetophenone (THAP), 3-hydroxypicolinic acid (3-HPA), and dithranol. The selection of an appropriate matrix for each sample is crucial in MALDI-MS analyses. For protein analyses, SA 7 , 8 or DHB 8 are effective, while peptide analyses commonly use CHCA 8 , 9 or DHB. 8 Carbohydrate analyses use DHB exclusively. 8 , 10 - 12 Oligonucleotide analyses require either THAP 13 or 3-HPA 14 as the matrix. Lipids are analyzed using a DHB 8 , 15 or dithranol 16 matrix, while synthetic polymers are commonly analyzed using dithranol. 17 Recently, 2-nitrophloroglucinol was introduced as a new matrix material that enhances multiply charged protein peaks in MALDI-MS analyses. 18 , 19 Figure 2 shows the chemical structures of several MALDI matrix materials. If the identity of the sample is completely unknown, then DHB is a good starting matrix, since it suits a wide variety of samples as mentioned above.
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Chemical structures of common matrix materials used in MALDI analyses.
There are two major drawbacks in MALDI-MS analyses using the above mentioned common matrices. First, there is always interference in the low m/z region, e.g ., < m/z 500, due to matrix fragments or its cluster peaks. DHB yields peaks at m/z 137.0 [DHB - H 2 O + H] + , 154.0 [DHB·] +• , 155.0 [DHB + H] + , 177.0 [DHB + Na] + , 199.0 [DHB - H + 2Na] + , and 273.1 [2DHB - H 2 O - OH] + . 20 CHCA provides fragment and cluster peaks at m/z 379.1 [2CHCA + H] + , 234.0 [CHCA - H + 2Na] + , 212.0 [CHCA + Na] + , 190.0 [CHCA + H] + , 172.0 [CHCA - H 2 O + H] + , and 146.1 [CHCA - CO 2 + H] + . 21 Therefore, MALDI is useful primarily in the high m/z region. The above peaks may, however, be used as internal standards in the low m/z region, thereby increasing the mass accuracy of MALDI analyses. The second drawback is the possibility of inhomogeneity in the distribution of matrix within a given MALDI spot. This often leads to poor reproducibility and poor quantitative results. These disadvantages can, in part, be overcome by developing novel matrix systems or by including various additives to the matrix as discussed below.
Novel matrix systems
To mitigate the effects of sample spot inhomogeneity, binary matrices that include two matrix materials, such as CHCA/DHB, 22 , 23 DHB/aminopyrazine, 24 CHCA/3-HPA for phosphopeptides, 25 and DHB/CHCA or DHB/SA for glycans and glycoproteins, 26 have been developed. Recently, a combination of three materials (DHB, CHCA, and SA) in a single matrix was used to improve performance in analyses of polyethylene glycol (PEG) polymers. 27
The combination of an organic acid matrix with a base often yields a viscous liquid known as an ionic liquid. Such materials have more homogeneous surfaces than the crystalline solid surface of an organic acid matrix. Ionic liquids have been used as alternative matrix materials. 28 , 29 For example, an ionic liquid composed of DHB and aniline has been used in carbohydrate analyses 30 and various ionic liquid matrices have been used to characterize bacteria. 31 Recently, the combination of two matrix materials (DHB and CHCA) with a base (piperidine) and trifluoroacetic acid was found beneficial in phospholipid analyses in both positive and negative ion mode. 32
Laser desorption/ionization in the absence of a matrix material has also been evaluated, using an etched porous silicon surface to trap the analyte. This method is called desorption ionization on silicon (DIOS). 33 Other alternatives to conventional organic acid matrices include carbon nanotubes, 34 graphite, 35 pencil lead, 36 , 37 gold nanoparticles, 38 , 39 graphene, 40 graphene oxide, 41 mildly oxidized graphene, 42 and wood charcoal. 43
A small amount of additive is often used to enhance MALDI signals. Examples include the addition of phosphoric acid to a DHB matrix to improve the intensity of phosphopeptide peaks 44 or non-phosphopeptide peaks, 45 the addition of serine to a CHCA matrix to eliminate alkali-metal ion adducts, 46 the addition of a small amount of detergent (Tween 80) to a CHCA matrix to reduce surface tension and enhance the uniformity of protein analyte distribution, 47 and the addition of nitrilotriacetic acid to a CHCA matrix to enhance peptide signals. 48 In order to be protonated and detected in positive ion mode MS, samples that do not contain basic residues require a cation additive such as Na + , Cs + , or Ag + . 49 - 51
Sample preparation methods
Several different methods are used to load sample and matrix materials onto MALDI plates. The oldest and most commonly used method is the dried droplet method, in which small portions of the matrix and analyte solutions are mixed. The mixture is then deposited on the MALDI plate and dried. 52 In the two-layer, or over-layer, method, a matrix solution is applied first to the MALDI plate, followed by the deposition of a mixture of matrix and analyte solutions. 53 In the fast evaporation or thin-layer method, matrix and sample solutions are kept separate. A thin layer of matrix crystal is prepared on a MALDI spot. The analyte solution is then deposited on top of the matrix. 54 The sample can also be sandwiched between two matrix layers. In this method, a sample droplet is applied on top of a fast-evaporated matrix spot followed by the deposition of a second layer of matrix material. 55 Several research articles have compared the various means of preparing MALDI samples. 54 - 56 Recently, an active drying technique was introduced wherein the final analyte/matrix drop on the MALDI plate is dried rapidly in a vacuum chamber or under an air flow in order to increase crystal homogeneity and sensitivity. Compared to passive drying, this active drying technique results in a uniform, disordered polymorph and can provide 5- to 10-fold greater sensitivity in MALDI-MS analyses of carbohydrates in a DHB matrix. 57
It is important to identify a suitable matrix and sample preparation method for an investigation. Padliya and Wood 56 compared different matrices and different sample preparation methods in MALDI-MS analyses of tryptically digested protein samples. They found that DHB generally provided higher sequence coverage than other matrices, while no single sample preparation method was exclusively better than the other methods. Kussmann et al . showed that there is no universal sample preparation method that is applicable to all analytes. 54 One might need to try several sample preparation methods in order to determine which one works best for one’s purposes.
Due to their inherently easy operation, fast analysis times, high mass accuracy, and high mass resolution, MALDI-MS analyses are popular in various fields and expected to increase in popularity. To fulfill the demand, various efforts have been exerted in MALDI sample preparation using neat matrices, new combinations of matrices, and suitable additives. These efforts are expected to continue to improve MALDI signals and provide reproducible results. A great deal of effort must usually be expended to analyze an uncharacterized sample, since each analyte may require a different matrix or sample preparation method. Generation of a universal matrix system would greatly benefit the researchers in the field of MALDI-MS. MALDI imaging mass spectrometry (IMS) is also gaining popularity. In MALDI-IMS, the spatial distribution of molecules of interest can be visualized without labeling and can result in information comparable to that obtained from traditional histology. 58 MALDI-MS spectra are known to be irreproducible. Recently, sample inhomogeneity and variation in MALDI plume temperatures were identified as two factors responsible for spectral irreproducibility. 59 Reproducible quantitative mass spectra were obtained at a fixed initial plume temperature, achieved using a total ion current-control method. 60 It is expected that much more effort will be focused on improving the reproducibility of MALDI-MS analyses and thereby providing reliable quantitative information.
This work was supported by research fund of Chungnam National University.
Jaskolla T. , Karas M. 2011 J. Am. Soc. Mass Spectrom. 22 976 -    DOI : 10.1007/s13361-011-0093-0
Zenobi R. , Knochenmuss R. 1998 Mass Spectrom. Rev. 17 337 -
Zavalin A. , Yang J.H. , Caprioli R. 2013 J. Am. Soc. Mass Spectrom. 24 1153 -    DOI : 10.1007/s13361-013-0638-5
McLean J. A. , Russell W. K. , Russell D. H. 2003 Anal. Chem. 75 648 -    DOI : 10.1021/ac026029y
Karas M. , Bachmann D. , Hillenkamp F. 1985 Anal. Chem. 57 2935 -    DOI : 10.1021/ac00291a042
Tanaka K. , Waki H. , Ido Y. , Akita S. , Yoshida Y. , Yoshida T. , Matsuo T. 1988 Rapid Commun. Mass Spectrom. 2 151 -    DOI : 10.1002/rcm.1290020802
Han H. , Nho S. , Lee A. , Kim J. B. 2010 Korean Chem. Soc. 31 1527 -    DOI : 10.5012/bkcs.2010.31.6.1527
Marvin L. F. , Roberts M. A. , Fay L. B. 2003 Clin. Chim. Acta 337 11 -    DOI : 10.1016/j.cccn.2003.08.008
Wiangnon K. , Cramer R. 2015 Anal. Chem. 87 1485 -    DOI : 10.1021/ac504412p
Ozcan S. , An H. J. , Vieira A. C. , Park G. W. , Kim J. H. , Mannis M. J. , Lebrilla C. B. 2013 J. Proteome Res. 12 1090 -    DOI : 10.1021/pr3008013
Park E. , Yang H. , Kim Y. , Kim J. 2012 Food Chem. 134 1658 -    DOI : 10.1016/j.foodchem.2012.03.069
Harvey D. J. 1999 Mass Spectrom. Rev. 18 349 -
Zhu Y. F. , Chung C. N. , Taranenko N. I. , Allman S. L. , Martin S. A. , Haff L. , Chen C. H. 1996 Rapid Commun. Mass Spectrom. 10 383 -
Wu K. J. , Steding A. , Becker C. H. 1993 Rapid Commun. Mass Spectrom. 7 142 -    DOI : 10.1002/rcm.1290070206
Wei Y. B. , Zhang Y. Y. , Lin Y. , Li L. , Liu J. A. , Wang Z. P. , Xiong S. X. , Zhao Z. W. 2015 Analyst 140 1298 -    DOI : 10.1039/C4AN01964D
Le C. H. , Han J. , Borchers C. H. 2012 Anal. Chem. 84 8391 -    DOI : 10.1021/ac301901s
Belu A. M. , DeSimone J. M. , Linton R. W. , Lange G. W. , Friedman R. M. 1996 J. Am. Soc. Mass Spectrom. 7 11 -    DOI : 10.1016/1044-0305(95)00589-7
Trimpin S. , Ren Y. , Wang B. , Lietz C. B. , Richards A. L. , Marshall D. D. , Inutan E. D. 2011 Anal Chem 83 5469 -    DOI : 10.1021/ac2007976
Lee J. , Hong J. , Kim T. , Kim J. 2013 Mass Spectrom. Lett. 4 21 -    DOI : 10.5478/MSL.2013.4.1.21
Yang H. -J. , Lee A. , Lee M. -K. , Kim W. , Kim J. 2010 B. Korean Chem. Soc. 31 35 -    DOI : 10.5012/bkcs.2010.31.01.035
Laugesen S. , Roepstorff P. 2003 J. Am. Soc. Mass Spectrom. 14 992 -    DOI : 10.1016/S1044-0305(03)00262-9
Signor L. , Erba E. B. 2013 J. Vis. Exp. 7
Hashir M. A. , Stecher G. , Bonn G. K. 2008 Rapid Commun. Mass Spectrom. 22 2185 -    DOI : 10.1002/rcm.3602
Zhou L. H. , Kang G. Y. , Kim K. P. 2009 Rapid Commun. Mass Spectrom. 23 2264 -    DOI : 10.1002/rcm.4139
Lastovickova M. , Chmelik J. , Bobalova J. 2009 Int. J. Mass Spectrom. 281 82 -    DOI : 10.1016/j.ijms.2008.12.017
Hong J. , Kim T. , Kim J. 2014 Mass Spectrom. Lett. 5 49 -    DOI : 10.5478/MSL.2014.5.2.49
Tholey A. , Heinzle E. 2006 Anal. Bioanal. Chem. 386 24 -    DOI : 10.1007/s00216-006-0600-5
Crank J. A. , Armstrong D. W. 2009 J. Am. Soc. Mass Spectrom. 20 1790 -    DOI : 10.1016/j.jasms.2009.05.020
Snovida S. I. , Rak-Banville J. M. , Perreault H. 2008 J. Am. Soc. Mass Spectrom. 19 1138 -    DOI : 10.1016/j.jasms.2008.04.033
Abdelhamid H. N. , Gopal J. , Wu H. -F. 2013 Analytica Chimica Acta 767 104 -    DOI : 10.1016/j.aca.2012.12.054
Shanta S. R. , Zhou L. H. , Park Y. S. , Kim Y. H. , Kim Y. , Kim K. P. 2011 Anal. Chem. 83 1252 -    DOI : 10.1021/ac1029659
Wei J. , Buriak J. M. , Siuzdak G. 1999 Nature 399 243 -    DOI : 10.1038/20400
Xu S. Y. , Li Y. F. , Zou H. F. , Qiu J. S. , Guo Z. , Guo B. C. 2003 Anal. Chem. 75 6191 -    DOI : 10.1021/ac0345695
Sunner J. , Dratz E. , Chen Y. -C. 1995 Anal. Chem. 67 4335 -    DOI : 10.1021/ac00119a021
Black C. , Poile C. , Langley J. , Herniman J. 2006 Rapid Commun. Mass Spectrom. 20 1053 -    DOI : 10.1002/rcm.2408
Langley G. J. , Herniman J. M. , Townell M. S. 2007 Rapid Commun. Mass Spectrom. 21 180 -    DOI : 10.1002/rcm.2827
McLean J. A. , Stumpo K. A. , Russell D. H. 2005 J. Am. Chem. Soc. 127 5304 -    DOI : 10.1021/ja043907w
Su C. -L. , Tseng W. -L. 2007 Anal. Chem. 79 1626 -    DOI : 10.1021/ac061747w
Dong X. , Cheng J. , Li J. , Wang Y. 2010 Anal. Chem. 82 6208 -
Liu J. Y. , Liu Y. , Gao M. X. , Zhang X. M. 2012 J. Am. Soc. Mass Spectrom. 23 1424 -    DOI : 10.1007/s13361-012-0400-4
Liu Q. , Cheng M. T. , Jiang G. B. 2013 Chem.-Eur. J. 19 5561 -    DOI : 10.1002/chem.201203791
Lee S. , Kim J. , Yang H. -J. , Shin S. , Hong J. , Kim J. 2010 Mass Spectrom. Lett. 1 33 -    DOI : 10.5478/MSL.2010.1.1.033
Kjellström S. , Jensen O. N. 2004 Anal. Chem. 76 5109 -    DOI : 10.1021/ac0400257
Park S. , Kim T. , Lee J. , Seo M. , Kim J. 2013 Rapid Commun. Mass Spectrom. 27 842 -    DOI : 10.1002/rcm.6508
Nishikaze T. , Takayama M. 2007 Rapid Commun. Mass Spectrom. 21 3345 -    DOI : 10.1002/rcm.3219
Brinkworth C. S. , Bourne D. J. 2007 J. Am. Soc. Mass Spectrom. 18 102 -    DOI : 10.1016/j.jasms.2006.09.003
Kim J. S. , Kim J. Y. , Kim H. J. 2005 Anal. Chem. 77 7483 -    DOI : 10.1021/ac051152l
Choi S. S. , Ha S. H. 2006 B. Korean Chem. Soc. 27 1243 -    DOI : 10.5012/bkcs.2006.27.8.1243
Laremore T. N. , Linhardt R. J. 2007 Rapid Commun. Mass Spectrom. 21 1315 -    DOI : 10.1002/rcm.2964
Choi S. -S. , Ha S. -H. 2008 Macromol. Res. 16 108 -    DOI : 10.1007/BF03218838
Karas M. , Hillenkamp F. 1988 Anal. Chem. 60 2299 -    DOI : 10.1021/ac00171a028
Dai Y.Q. , Whittal R. M. , Li L. 1999 Anal. Chem. 71 1087 -    DOI : 10.1021/ac980684h
Kussmann M. , Nordhoff E. , RahbekNielsen H. , Haebel S. , RosselLarsen M. , Jakobsen L. , Gobom J. , Mirgorodskaya E. , KrollKristensen A. , Palm L. , Roepstorff P. 1997 J. Mass Spectrom. 32 593 -
Kemptner J. , Marchetti-Deschmann M. , Mach R. , Druzhinina I. S. , Kubicek C. P. , Allmaier G. 2009 Rapid Commun. Mass Spectrom. 23 877 -    DOI : 10.1002/rcm.3949
Padliya N. D. , Wood T. D. 2008 Anal. Chim. Acta 627 162 -    DOI : 10.1016/j.aca.2008.05.059
Williams T. I. , Saggese D. A. , Wilcox R. J. , Martin J. D. , Muddiman D. C. 2007 Rapid Commun. Mass Spectrom. 21 807 -    DOI : 10.1002/rcm.2904
Aichler M. , Walch A. 2015 Lab. Invest. 95 422 -    DOI : 10.1038/labinvest.2014.156
Ahn S. H. , Park K. M. , Moon J. H. , Lee S. H. , Kim M. S. 2015 Rapid Commun. Mass Spectrom. 29 745 -    DOI : 10.1002/rcm.7157
Ahn S. , Park K. , Bae Y. , Kim M. 2013 J. Am. Soc. Mass Spectrom. 24 868 -    DOI : 10.1007/s13361-013-0612-2