Received date: March 08, 2012; Accepted date: March 29, 2012; Published date: March 31, 2012
Citation: Liu JA, Xiong L, Zhang S, Wei JC, Xiong SX (2012) C60 Fluorine Derivative as Novel Matrix for Small Molecule Analysis by MALDI-TOF MS. Metabolomics S1:002. doi: 10.4172/2153-0769.S1-002
Copyright: © 2012 Liu JA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an efficient tool for large molecule analysis including proteins, carbohydrates, polymers etc., with the advantages of high sensitivity, high throughput and high speed. However, the traditional matrices, such as α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), fail to detect the compounds with molecular weight below 500 Da due to the interference from matrix background. In this paper, we used C60 fluorine derivative as matrix for small molecule analysis by MALDI-TOF MS. With the successful detection of various small molecules from saccharides, plant extracts, fatty acids, amino acids to pesticides, C60 derivative is reported here for the first time as a novel and effective MALDI matrix for small molecule analysis in metabolism and compound characterization areas.
C60 Fluorine derivative; Matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry; Matrix; Small molecule
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was introduced in 1980s by Tanaka and Hillenkamp [1,2]. It has been considered as a convenient instrument to study proteins , oligosaccharides  and synthetic macromolecules , because of its high speed, high throughput, high stability, etc. However, it couldn't analyze small molecules (< 500 Da) efficiently with the use of traditional matrices, such as α-cyano- 4-hydroxycinnmaic acid (CHCA) and 2, 5-Dihydroxy benzonic acid (DHB), due to the strong matrix-related background noise generated in the low-mass region, which interferes with the analysis of the target analytes.
Many methods have been reported to conquer this problem. Desorption/ionization on porous silicon  with silicon materials, e.g. silicon nanocavity  and silicon nanowires , and non-silicon substances such as porous aluminum  and zinc oxide nanoparticles  has been reported working efficiently for small molecule analysis. Alternatively, utilization of matrix suppression effect [11,12] could reduce the matrix-induced background substantially. On the other hand, the most straightforward way was to explore new matrices, such as carbon nanotube , graphite [14,15] and high-mass molecules [16,17]. Recently, graphene  was utilized for the first time as a matrix for low molecular weight compounds, including polar compounds and nonpolar compounds in positive ion mode. And also it can be used as an adsorbent for the solid-phase extraction of squalene could improve greatly the detection limit. In negative ion mode, graphene flakes  was used as matrix and deprotonated monomeric species with significant reduction of matrix interference were detected for the analysis of peptides, amino acids, fatty acids as well as nucleosides and nucleotides. Better sensitivity and reproducibility were achieved in the analyses with negative ion mode than positive ion mode .
In this report, we focus on another interesting group of high-mass molecules, fluorine derivatives of C60, as a novel MALDI matrix for small molecule analysis. The hydrophobic property of the compound facilitates sample converging and further improves the detection limits of analytes. Using this novel matrix, clean MALDI spectra of various types of small molecules including sucrose, glucose, gibberellic acid (GA3), palmitic acid, quercetin, vitamin C, leucine, dipeptide asp-phe, metalaxyl and carbofuran were obtained successfully in positive or negative ion mode. With the application of C60 fluorine derivative as matrix, MALDI-TOF MS could be considered as an efficient tool for high speed analysis of low molecular weight compounds, especially for metabolism research, environmental compound screening and natural product characterization.
The C60 fluorine derivative supplied by the Key Laboratory of Organic Solids of Institute of Chemistry has been characterized with IR, NMR, MS, etc. Palmitic acid and metalaxyl were from Sigma (St. Louis, MO, USA). Gibberellic acid (GA3) was purchased from Acros (New Jersey, USA). CHCA, leucine and carbofuran were from Aldrich (Milwaukee, WI, USA).
Samples and matrices were dissolved in solution of 50:50 acetonitrile/water (v/v) respectively. Then, 1 μL matrix and 1 μL sample solution were mixed together prior to the sample analysis.
All mass spectrometry experiments were performed on an Autoflex III MALDI-TOF mass spectrometer (Bruker Daltonics) equipped with a 355 nm nitrogen laser. Prior to experiment, the instrument was calibrated using external standard calibration method.
Small molecule analytes and matrix were dissolved in methanol separately, mixed and spotted on the SCOUT MTP 384 MALDI target plate. After solvent evaporation and crystallization, the target plate was placed onto the tray and inserted into the mass spectrometer for analysis. The mass spectra were acquired in reflectron mode with an acceleration voltage of 19 kV. The laser frequency was 10 Hz. The laser power was 40 - 90% of the maximum. Usually 100-300 laser shots were accumulated for each spectrum.
The structure and hydrophobicity of C60 fluorine derivative
Since the discovery of Fullerene C60 in 1985 , a variety of studies on it have been conducted. Fullerenes consist of 20 hexagonal and 12 pentagonal rings as the basis of icosahedra symmetry closed cage structure, Each carbon atom is bonded to three others and is sp2 hybridized, the inner and outer surface are covered with a sea of π electrons, which not only absorb laser power of MALDI source, but also transfer energy efficiently to neighboring molecules. In this work, we used C60 derivative depicted in Figure 1a as an ideal matrix for the analysis of low molecular weight compounds in MALDI-TOF MS, without generating background signal in the low mass range. It is also worth noting that the high hydrophobicity of C60 derivatives helps increase the surface tension of the sample droplet during MALDI plate spotting process (Figure 1b). Therefore, analytes in each MALDI sample spot were focused to a smaller area on the plate surface. The sample concentration per laser activated area is efficiently increased to provide better sensitivity of MS analysis.
The comparison of traditional MALDI matrices and C60 derivatives
First of all, we compared the MALDI MS results of small molecules analysis using traditional matrices and C60 derivative. Glucose was used as the test sample in positive ion mode. Glucose is an important monosaccharide, but very difficult to get ionized, because it is lack of benzyling ring or any conjugate structures to absorb energy. When analyzed using DHB matrix (commonly used matrix for saccharides), a lot of intense background peaks formed from DHB were observed in the MS spectrum (Figure 2a). The MS signal of the target glucose could hardly be identified because of the high background noise signals and the ionization suppression effects induced by matrix peaks. On the other hand, a clear spectrum was obtained using C60 derivative as matrix (Figure 2b). In the spectrum, the sodium and potassium adduct ions of glucose were detected with high intensities at m/z 203.1 for [M+Na] + and m/z 219.0 for [M+K]+. There is no matrix related signal shown in the spectrum.
In negative ion mode, gibbrellic acid (GA3) was utilized as test sample. GA3 is a 19 carbon diterpenoid compound, which serves as an important phytohormone and plays an essential role in seed germination, cell division and elongation, flowering and so on [21,22]. When using CHCA as matrix in negative ion mode, only weak signal of GA3 was found with interferences of matrix peaks from CHCA (Figure 3a). However, clear spectrum with high signal intensity of GA3 deprotonated ion was obtained using C60 derivatives matrix (Figure 3b), detected at m/z 345.1.
The results indicated that C60 fluorine derivative was effective MALDI matrix for low-molecular weight analytes in both positive and negative ion mode.
Application to different types of small molecules
Oligosaccharide is difficult to be detected by MALDI-TOF MS because of its low ionization efficiency and low molecular weight . Except monosaccharide, such as glucose, C60 derivative was also successfully used as MALDI matrix for disaccharide detection. The MALDI spectrum of sucrose is shown in Figure 4a, with the presences of sodium adduct ion at m/z 365.1 and potassium adduct ion at m/z 381.1. This result was comparable with the sucrose MALDI analysis using modified mesoporous material SBA-15 as assisted matrix .
Different amino acid and small peptides were also analyzed by MALDI-TOF MS when using C60 derivatives as matrix. Figure 4b shows that leucine was detected in negative ion mode in the form of [MH] - at m/z 130.1. Figure 4c shows the MS spectrum of dipeptide aspphe in positive ion mode, and the peaks in the spectrum were adduct ions, [M+Na]+ at m/z 301.1 and [M+K]+ at m/z 319.1, respectively.
Except phytohormone GA3 discussed previously, the new matrix was also applied to study other plant extracts. Flavonoids, such as quercetin and luteolin, were able to be detected in both positive ion mode and negative ion mode, because they were weak acids. Figure 4d shows the spectrum of quercetin in positive ion mode. Protonated ion and sodium adduct ion were obtained, at m/z 303.0 and m/z 325.0, respectively. In negative ion mode, deprotonated ions [M-H]- at m/z 301.0 was detected (data not shown). MALDI spectrum of vitamin C in negative ion mode is shown in Figure 4e. In the spectrum, the deprotonated analyte signal was detected clearly with high signal intensity at m/z 175.0.
Fatty acid is a kind of saturated or unsaturated acids with long aliphatic chains. Because of lacking phenyl ring and its small molecular mass, it is difficult to be analyzed by MALDI-TOF MS. It was reported in the previous publication that high molecular-weight matrix, (mesotetrakis (pentafluorophenyl) porphyrin) was used for the determination of the fatty acid composition of vegetable oils in positive ion mode . In our study, fatty acid was successfully detected by MALDITOF MS in negative ion mode using C60 derivative matrix. Figure 4f shows that palmitic acid was analyzed in the form of deprotonated ions [M-H]- at m/z 255.2, which was similar to those using proton sponge (1,8-bis(dimethyl-amino)naphthalene, DMAN) as matrix [26,27].
Detection of pesticides and their metabolites in vegetablesm fruits extracts and human plasma, urine samples has become an important and popular application area of mass spectrometry which has attracted world-wide attentions nowadays. The novel matrix we explored also works well for the pesticide analysis. Figure 5a shows the MALDI spectrum of metalaxyl in the presence of sodium adduct ion [M+Na]+ at m/z 302.1 with high intensity. While Figure 5b shows the MALDI spectrum of carbofuran, with its sodium adduct ion [M+Na]+ at m/z 224.1 detected in positive ion mode.
C60 fluorine derivative was reported here as a novel MALDI matrix for small molecule analysis in both positive and negative ion mode, without bringing obvious matrix related background signal in the low mass range. Moreover, its strong hydrophobicity effectively enhances the sample focusing during the MALDI sample spot preparation thereby further improving the detection limit of MALDI MS analysis. With the successful detection of various types of small molecules including oligosaccharides, plant extracts, amino acids and small peptides, fatty acids, and pesticides, all with molecular weights lower than 500 Da, the application of C60 fluorine derivative as matrix opens a new window for high-speed, high-throughput small molecule detection/screening by using MALDI MS, especially in metabolism and natural compound characterization areas.
This work was financially supported by NSFC (No.90717120 and 20435030), MOST (No.2007CB714504 and 2009IM031200) and CAS (KJCX2-YW-H11).