EP2166560A1 - Maldi-Matrizen - Google Patents

Maldi-Matrizen Download PDF

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Publication number
EP2166560A1
EP2166560A1 EP08016619A EP08016619A EP2166560A1 EP 2166560 A1 EP2166560 A1 EP 2166560A1 EP 08016619 A EP08016619 A EP 08016619A EP 08016619 A EP08016619 A EP 08016619A EP 2166560 A1 EP2166560 A1 EP 2166560A1
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Prior art keywords
matrix
maldi
compounds
analysis
analytes
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EP08016619A
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English (en)
French (fr)
Inventor
Ales Dr. Svatos
Rohit Shroff
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Priority to EP08016619A priority Critical patent/EP2166560A1/de
Priority to PCT/EP2009/006838 priority patent/WO2010031588A2/en
Publication of EP2166560A1 publication Critical patent/EP2166560A1/de
Withdrawn legal-status Critical Current

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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
    • H01J49/161—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission using photoionisation, e.g. by laser
    • H01J49/164—Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI]

Definitions

  • the present invention relates to novel MALDI matrices for the analysis of low-molecular-weight compounds in both positive and negative ion mode.
  • the present invention relates to the rational selection of the MALDI matrices for predetermined analytes, a process which has remained empirical since the invention of the technique.
  • the present invention provides a new, fast and high-throughput method for analyzing low-molecular-weight compounds by MALDI analysis as well as systems therefore.
  • MALDI Matrix-Assisted Laser Desorption/Ionization
  • MALDI-MS finds far reaching applications in various fields including, but not exclusive, proteomics, nucleic acid analysis, analysis of lipids, glycans and polymers.
  • biomarker detection using imaging MALDI MS has been developed.
  • MALDI MS only a very restricted number of matrix compounds are used; these were discovered very early during development of the MALDI technique.
  • Typical matrix compounds include ⁇ -Cyano-4-hydroxycinnamic acid, 2,5-Dihydroxbenzoic acid (DHB) which are suitable for peptide, protein, lipid and oligosaccharide analysis and sinapinic acid for protein analysis.
  • DVB 2,5-Dihydroxbenzoic acid
  • DIOS-MS porous silicon-mass spectrometry
  • the analyte to be investigated is typically co-crystallized with the matrix whereby the matrix is used in a 100 to 100,000 times molar excess to the analyte.
  • the co-crystallization of the sample takes place on the sample support, thus, incorporating the analyte into the matrix.
  • successful co-crystallization requires a matrix to analyte ratio of about 5000 fold for peptide analysis.
  • an object of the present invention is to provide matrices useful for analyzing low-molecular-weight compounds by matrix assisted laser desorption/ionization (MALDI) analysis in negative as well as positive ion mode.
  • Another object of the present invention is directed to methods for analyzing low-molecular-weight compounds, in particular, of allowing quantitative analysis of said low-molecular-weight compounds.
  • Another object of the present invention relates to a method for rational selection of the appropriate MALDI matrices for the analysis of predetermined analytes.
  • the present invention relates to a matrix for matrix assisted laser desorption/ionization (MALDI) analysis of low-molecular-weight acidic compounds in negative ion mode wherein said matrix compounds are of general formula I wherein
  • the present invention relates to a matrix for MALDI analysis in positive ion mode allowing detection of low-molecular-weight basic compounds, said matrix is a compound of the general formula IV R 5 -(Z-H) n (IV)
  • Another aspect of our invention relates to the optimization of matrix to analyte ratio for analysis of low-molecular-weight compounds.
  • a huge molar excess of the matrix is used for peptide and protein analysis.
  • equimolar concentrations of matrix to analyte is optimal for maximal analyte signal and complete matrix suppression by comparing different matrix to analyte ratios from 0.1:1 to 100:1.
  • the present invention relates to a system for the analysis of low-molecular-weight compounds with MALDI comprising the matrix according to the present invention and, optionally, a sample support.
  • the present invention relates to novel and rational matrix development for Matrix Assisted Laser Desorption/Ionization (MALDI) analysis of low-molecular-weight compounds.
  • MALDI Matrix Assisted Laser Desorption/Ionization
  • the present invention relates on one hand to a matrix for MALDI analysis in a negative ion mode and, on the other hand, to a matrix for MALDI analysis in positive ion mode. Both allowing detection of various analytes, in particular low-molecular-weight compounds.
  • the present invention relates to rationalization of the MALDI matrix selection process depending on the polarity of analysis and the polarity of the compounds to be studied. This represents a significant advance since matrix selection, which is the heart of the MALDI process, has remained an empirical approach since the birth of the technique.
  • the matrix compounds according to the present invention are of general formula I wherein
  • aliphatic group refers to carbon atoms joint together in a straight-chain or branched chain which may be substituted, including alkanes, alkenes and alkynes. Substituents are typical substituents of carbon atom groups, like hydroxy groups, carboxylate groups, nitrogen containing groups, sulphur containing groups, oxygen containing groups and halogens.
  • alicyclic group refers to carbon atoms forming a nonaromatic ring system which may be substituted. Substituents of the alicyclic group include hydroxy groups, nitrogen containing groups, oxygen containing groups, sulphur containing groups, halogens, aliphatic groups etc.
  • aromatic group refers to groups having aromaticity, namely having a conjugated ring of unsaturated bonds or ion pairs of electrons and satisfying the Huckle's rule which states that the an aromatic system should have 4n + 2 electrons, where n is an integer ⁇ 0.
  • Said aromatic group includes aryl and heteroaryl groups whereby said heteroaryl groups may contain heteroatoms of N, O, B, P, or S.
  • the residue Y is a nitrogen atom. That is, preferably, the compound is a tertiary amino group, like dialkylamino group. Furthermore, n is preferably an integer of 1, 2, 3, or 4.
  • the matrices of general formula I for MALDI analysis in negative ion mode are matrix compounds of general formula II or III wherein
  • the residues R 1 and R 2 are independently selected from C 1 to C 12 aliphatic groups, like C 1 to C 12 alkane, in particular C 1 to C 6 alkane, like methyl, ethyl, propyl, butyl, pentyl or hexyl.
  • Particular preferred matrix compounds for MALDI analysis in negative ion mode are 1,8-bis(dimethylamino)naphthalene (DMAN), N,N-dimethylaniline, or aniline.
  • DMAN 1,8-bis(dimethylamino)naphthalene
  • aniline N,N-dimethylaniline
  • the matrix for MALDI analysis in negative ion mode is preferably characterized in that the pKa of matrix protonation is above 3, likely above 5, preferably above 11 and simultaneously pKa of deprotonation of residues R 1 , R 2 or R 3 bound to Y of the matrix, namely, the central N or P atom, is above 35, likely above 40, preferably above 50.
  • pka is defined as the negative logarithm of the acid dissociation constant Ka.
  • matrix compounds of general formula IV are used.
  • the residue Z is SO 3 and n is an integer of 1, 2, 3, or 4, in particular, 1.
  • the aromatic group is preferably a mono or bicyclic aromatic group, like a phenyl group or naphthalene group.
  • Said aromatic groups may be aryl groups or heteroaryl groups, preferably, aryl groups are present.
  • the substituent R 5 is preferably a mono or bicyclic aromatic group whereby said aromatic group is substituted with at least one substituent R 6 whereby R 6 is selected from a hydroxy group, an aliphatic group, an alicyclic group or an aryl group.
  • the aromatic group R 5 is not substituted or has substituents which will enhance the acidity of the matrix compounds through inductive and mesomeric effects without themselves having an exchangeable acidic proton or a basic functional group capable of protonation.
  • the matrix for MALDI analysis in positive ion mode is preferably characterized in that the pKa of matrix protonation is below -12, like below -20, preferably below -25 and simultaneously pKa of deprotonation of matrix Z-H group is above 35, like above 40, preferably above 50.
  • the matrix compounds according to the present invention are particularly useful for the analysis of low-molecular-weight compounds using MALDI.
  • the term "low-molecular-weight compounds” refers to compounds of ⁇ 2000 Daltons.
  • the method allows analyzing compounds below 1000 Daltons, like below 700 Daltons, preferably, below 500 Daltons.
  • Typical low-molecular-weight analytes include fatty acids, amino acids, fatty acid-amino acids conjugates, plant and animal hormones, vitamins, short peptides, aliphatic, cyclic and aromatic acids including but not exclusive very volatile acids like trifluoroacetic acid and trichloroacetic acid,
  • analytes to be studied using MALDI analysis in positive ion mode include basic low-molecular-weight analytes like extremely volatile bases like triethylamine, short and long chain aliphatic, cyclic and aromatic bases.
  • the present invention relates in a further aspect to a method for analysing low-molecular-weight compounds containing acidic function(s) as well as basic function(s) in a range as low as one picomole or even in the femtomole range.
  • the present invention provides a new possibility for analyzing said low-molecular-weight compounds, like biologically significant markers for various purposes using MALDI analysis with high-sensitivity and specificity.
  • the matrix itself have no peaks in the spectrum and, additionally, no peaks arising from neutral losses of water or carbon dioxide are observed.
  • the matrix compounds according to the present invention are particularly useful for analyzing said low-molecular-weight compounds.
  • One of the representative classes of said low-molecular-weight compounds include fatty acids.
  • the present invention allows the analysis of all types of fatty acids, saturated as well as unsaturated fatty acids which was not possible before.
  • Fatty acids are important biomolecules which have been studied extensively as biologically significant markers for diagnosing infectious diseases, exploring organismal response to environmental factors and for taxonomic species classification.
  • the present invention is not only limited to fatty acid analysis, which by itself is a significant advance, but also encompasses other chemically diverse analytes including short peptides, amino acids, vitamins, plant and animal hormones, aliphatic cyclic and aromatic acids and even extremely small volatile acids and bases like trifluoroacetic (TFA) and trichloroacetic (TCA) acids, triethylamine (TEA) which were never thought to be amenable under conventional vacuum MALDI conditions.
  • TFA trifluoroacetic
  • TCA trichloroacetic
  • TAA triethylamine
  • Fig 1 describes the ion formation using the matrix compounds of the nature according to the present invention in the negative ion mode, specifically using DMAN.
  • DMAN belongs to the class of compounds called "proton sponges". The name comes from the ability of the compounds to "mop up” any available protons.
  • our theory of ion formation is that on mixing with acidic analytes even the weakly acidic proton on the -COOH group of the analytes is taken up by the DMAN, more specifically, it chelates between the two nitrogen atoms on DMAN forming a 2-electron 3 centre bond. This creates a stable salt/ion pair between the analyte and the matrix in solution and the charge state of the respective compounds is retained in the solid crystalline phase.
  • Another embodiment of the present invention relates to a method for selecting appropriate matrix compounds to be used for MALDI analysis of predetermined analytes.
  • Said analytes are predetermined in the fact that the acidity or basicity is known.
  • the method for selecting an appropriate MALDI matrix for the analysis of either acidic or basic analytes comprises the following steps:
  • the MALDI matrix compounds are selected on the parameters that none of substituents of the central Y atom, namely, P or N, of general formula I contain acidic hydrogen atoms having a pKa of deprotonation higher than 40 and, in addition, the pKa of the matrix is same or higher then pKa of an analyte.
  • the MALDI matrix is selected on the basis that the Z or R 5 groups of general formula IV do not contain a basic atom or group with pKa of protonation below -10 and in addition, the pKa of the matrix is same or lower then pKa of an analyte.
  • the pKa of the MALDI matrix and of the substituents R 1 , R 2 and R 3 as well as R 5 and R 6 are typically calculated using quantum mechanical calculation in gas and condensed phase for example as illustrated for the four bases in table 1.
  • the present invention relates to a method for analyzing analytes, in particular low-molecular-weight compounds, containing acidic function(s) by MALDI analysis comprising the step of
  • the method for analyzing compounds, in particular, low-molecular weight compounds, by MALDI comprise the step of
  • the wavelength of the laser is 337 or 355 nm, respectively.
  • a further aspect relates to a system for the analysis of analytes, in particular, of low-molecular-weight compounds with MALDI comprising the matrix compounds according to the present invention.
  • said system further comprises a sample support wherein the mixture of the matrix compounds with the analytes are deposited.
  • said system contains further components required for preparing the matrix compound/analyte mixture including solvents, instruction for use etc.
  • the present invention allows for quantification of an analyte in a sample by MALDI analysis using the matrix compounds according to the present invention.
  • the method for quantification comprises the steps of
  • a wide variety of analytes including extremely volatile compounds can be analyzed using the matrix compounds according to the present invention.
  • Palmitic, stearic, arachidic, oleic, linoleic, linolenic acids and DMAN were purchased from Sigma-Aldrich (St. Louis, MO, USA).
  • the peptides were purchased from Bachem (Bubendorf, Switzerland).
  • PEG 600 Sulfate was purchased from TCI (Antwerp, Belgium).
  • Alprostadil was purchased from Tocris Bioscience (Elisville, MO, USA).
  • HPLC-grade solvents, methanol, ethanol, acetone and chloroform were purchased from Roth (Karlsruhe, Germany).
  • Synthetic FACs were kindly provided by the Department of Bioorganic Chemistry and the regurgitate of Manduca sexta by the Department of Molecular Ecology, both at the Max Planck Institute for Chemical Ecology, Jena, Germany.
  • a MALDI micro MX mass spectrometer (Waters/Micromass, Manchester, UK) fitted with a nitrogen laser (337 nm, 4 ns laser pulse duration, max 330 ⁇ J per laser pulse, max 20 Hz repetition rate) was used in reflectron mode and negative polarity for data acquisition.
  • the instrument operated with voltages of 5 kV on the sample plate, 12 kV on the extraction grid, pulse and detector voltages of 1.95 kV and 2.35 kV, respectively.
  • the laser frequency was set to 5 Hz and energy was optimized for different analytes (fatty acids at 80 ⁇ J per pulse, peptides at 90 ⁇ J per pulse).
  • the extraction delay time was optimized to 150 ns.
  • PEG 600 sulfate was used to calibrate the mass spectrometer for a mass range of 100-1200 Th in the negative ion mode.
  • a mixture of PEG 200 and 600 was used for positive mode calibration.
  • the chemical identity of the FACs observed in the M. sexta regurgitate was confirmed by tandem mass spectrometry on an ion trap (LTQ) instrument (Thermo Fisher, San Jose, CA, USA) with an AP-MALDI source equipped with a solid-state Neodymium-Doped Yttrium-Aluminium-Garnet (Nd-YAG) laser (MassTech, Columbia, MD, USA) and running Target 6 (MassTech) and Excalibur v.2.0 (Thermo) software for data acquisition.
  • LTQ ion trap
  • DMAN as matrix compound and diverse low-molecular-weight compounds as analytes
  • Fig. 7 shows the deprotonated ions for Glu-Val-OH at m / z 245.0 ( Fig. 7a ), Phe-Phe-Phe-OH at m / z 458.1 ( Fig. 7b ) and Glu-Val-Phe-OH at m / z 392.1 ( Fig. 7c ).
  • Fig. 8a shows the averaged mass spectrum from 20 scans obtained from the spot. Almost all the peaks observed in the spectrum were found to be either fatty acids or fatty acid-glutamic acid conjugates ( Fig. 8a ). The identity of the peaks was confirmed by carrying out Collision-Induced-Dissociation (CID) experiments and by comparing the MS 2 spectra observed to those obtained from standard compounds ( Fig. 8b, c, d and e and Table 1).
  • CID Collision-Induced-Dissociation
  • SA stearic acid
  • pK 10.15 a representative medium strong acid
  • Fig. 10 strong dependency of stearate ion abundance on pK values of used bases was observed ( Fig. 10 ).
  • Fig. 10b no matrix ions were observed for N,N -dimethylaniline ( Fig. 10b , DMA) and surprisingly, for aniline ( Fig. 10d ).
  • the negative MALDI spectrum using 1,8-diaminonaphthalene shows, beside expected m / z 283 of stearate, copious matrix cluster ions obscuring both high and low-mass regions ( Fig. 10c ).
  • Stock solutions of two acids namely, TFA and Stearic acids were made at 500 pmol/ ⁇ l.
  • Two matrices, DMAN and DMA were made at different concentrations.
  • Individual matrices were mixed at different amounts with individual analytes at fixed amounts so as to have a plurality of molar ratios ranging from 0.02:1, 0.05:1, 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1, 5:1, and 10:1 to 100:1 (Matrix/Analyte).
  • MALDI-TOF measurements were made in negative ion mode for each mixture for each set of matrix/analyte mixtures, namely, for DMAN/TFA, DMAN/SA, DMA/TFA and DMA/SA at the above mentioned variable molar ratios.
  • a plot of the relative intensity of analyte ions (here, TFA, solid line and SA, dashed line) were made against MALDI matrix concentrations (DMAN in 11a and DMA in 11b) plotted for clarity as log2 [matrix]. It is clear from the figure 11a and b that maximum analyte signal was observed at the point of equimolarity.
  • This strategy could be used for quantifying a diverse group of analytes by simply providing a plurality of matrix/analyte molar ratios and determining the point of equimolarity.

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  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Engineering & Computer Science (AREA)
  • Plasma & Fusion (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
EP08016619A 2008-09-22 2008-09-22 Maldi-Matrizen Withdrawn EP2166560A1 (de)

Priority Applications (2)

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EP08016619A EP2166560A1 (de) 2008-09-22 2008-09-22 Maldi-Matrizen
PCT/EP2009/006838 WO2010031588A2 (en) 2008-09-22 2009-09-22 Maldi matrices

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102175752A (zh) * 2011-01-06 2011-09-07 中国科学院生态环境研究中心 一种有机质子碱基质辅助的全氟酸类物质高灵敏检测方法
JP2013130570A (ja) * 2011-11-21 2013-07-04 Yokohama City Univ タンパク質とリン酸化ペプチドのペプチド主鎖のＮ−Ｃα結合又はＣα−Ｃ結合の特異的切断方法

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103063730B (zh) * 2012-12-13 2015-01-07 中国科学院化学研究所 萘肼无机酸盐或萘肼有机酸盐在作为基质辅助激光解吸电离质谱中基质的应用
JP6156846B2 (ja) * 2014-03-04 2017-07-05 株式会社島津製作所 マトリックス支援レーザ脱離イオン化質量分析用マトリックス
JP6156845B2 (ja) * 2014-03-04 2017-07-05 株式会社島津製作所 マトリックス支援レーザ脱離イオン化質量分析用マトリックス
US9880147B2 (en) 2015-05-29 2018-01-30 Waters Technologies Corporation Free and total fatty acid determination using desorption ionization—mass spectrometry
CN116203119B (zh) * 2021-12-01 2024-02-06 生物岛实验室 一种用于小分子化合物检测的maldi-ms基质及检测方法

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102175752A (zh) * 2011-01-06 2011-09-07 中国科学院生态环境研究中心 一种有机质子碱基质辅助的全氟酸类物质高灵敏检测方法
JP2013130570A (ja) * 2011-11-21 2013-07-04 Yokohama City Univ タンパク質とリン酸化ペプチドのペプチド主鎖のＮ−Ｃα結合又はＣα−Ｃ結合の特異的切断方法

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WO2010031588A3 (en) 2010-08-26

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