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Isotope Labeling Strategies for Acylcarnitines Profile in Biological Samples by Liquid Chromatography-Mass Spectrometry Shangfu Li, Dan Gao, Chao Song, Chunyan Tan, and Yuyang Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05120 • Publication Date (Web): 12 Jan 2019 Downloaded from http://pubs.acs.org on January 14, 2019
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Analytical Chemistry
Isotope labeling strategies for acylcarnitines profile in biological samples by liquid chromatography-mass spectrometry Shangfu Li,†, ‡ Dan Gao,*,†,‡,§ Chao Song,†,‡ Chunyan Tan,*, †,‡ Yuyang Jiang†,|| †State
Key Laboratory of Chemical Oncogenomics, Graduate School at Shenzhen, Tsinghua University, Shenzhen, Guangdong 518055, China ‡National
& Local United Engineering Lab for Personalized Anti-tumor Drugs, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China §Key
Laboratory of Metabolomics at Shenzhen, Shenzhen 518055, China
||School
of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China
ABSTRACT: Acylcarnitines are closely related to many metabolic diseases and likely to be good biomarkers for clinical diagnosis. Studies on acylcarnitines may help deepen the understanding of their functions associated with disease processes. However, the diversity of their structures and lack of commercial standards made them difficult to be fully detected. In this work, a highly efficient isotope labeling strategy was developed to detect acylcarnitines in biological samples using liquid chromatography-mass spectrometry (LC-MS). A pair of reagents with or without deuterium labeling tags containing both a positive charge and a primary amine group were synthesized, which were used to label the carboxyl group of acylcarnitines. The reaction was performed under mild conditions to avoid hydrolysis of acylcarnitines. The reaction products had two positive charges, forming a double charge peak in MS spectra and with mass-to-charge ratio difference (Δm/z) of 4.0251 Da between the light- and heavy-labeled products. The feature made it possible to distinguish signals of acylcarnitines from other carboxyl metabolites with Δm/z of 8.0502 Da between their light- and heavy-labeled products. Based on the characteristics and the MS/MS spectra, a total of 108 acylcarnitines were discovered and identified in urine samples. Our established approach will be great helpful for the studies of diseases associated with acylcarnitines metabolism.
Acylcarnitines involve in many biological processes.1 They were key factors in regulating the balance of intracellular metabolism of substances and energy.2 Metabolic disorder of acylcarnitines were closely related to numerous diseases,3 such as genetic abnormalities in neonates,4 type I and type II diabetes5 and carcinomatosis.6 Some of acylcarnitines have been considered as potential biomarkers for clinical diagnosis.7 Due to the close relationship between acylcarnitines and different kinds of diseases, their researches have gained increasing attention in recent years. Therefore, to profile the acylcarnitines in biological samples may deepen our understanding of the functions of these compounds and related disease mechanisms, which will bring benefit to disease diagnosis and treatment. Many methods have been developed for the analysis of acylcarnitines. For sample preparation, derivatization strategies are often used, such as anhydrous n-butanol/HCl-based method.8 But acid-catalyzed reaction condition results in hydrolysis of the acylcarnitines, leading to inaccurate measurement.9 In recent years, non-derivatization methods combined with liquid chromatography-mass spectrometry (LCMS) have become popular for acylcarnitine detection.10-13 Due to the excellent separation ability and high sensitivity of LCMS, dozens of acylcarnitines could be analyzed simultaneously.14-16 By using highly selective scanning modes, such as selected reaction monitoring and parallel reaction monitoring, up to hundreds of acylcarnitines could be identified
in plasma, urine and tissue samples.17-19 These results provide significant reference value to annotation of acylcarnitines in biological samples. However, due to the diversity of acylcarnitines and lack of commercial standards, qualitative and quantitative analysis of acylcarnitines by non-derivatization methods still face challenges in the absence of sufficient evidence for structural confirmation. Some of acylcarnitine standards can be obtained by conjugating the corresponding carboxyl compounds with L-carnitine, but only a small fraction of currently known acylcarnitines can be synthesized because the carboxyl compounds are also facing the same fate of standard shortages. These problems limit the application of previously reported methods for the detection of acylcarnitines, especially for quantitative analysis. Therefore, the development of new approaches to efficiently and accurately identify acylcarnitines are still in urgent need. Isotope labeling methods have a wide range of applications for the selective identification of compounds containing specific functional groups, such as cis-diols, carboxyl, amine, and thiol groups.20-24 These compounds could be qualitatively or relative quantitatively analyzed depending on the characteristics of the isotope labeling reagents. All the acylcarnitines contain a carboxyl group which can also serve as an isotope labeling reaction site. However, there is few relevant report yet. In this work, an isotope labeling method for the nontargeted profiling of acylcarnitines in biological samples was developed through a mild, rapid, and quantitative derivatization
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Figure 1. The schematic illustration for the profiling of acylcarnitines. (A) The synthesis of isotope labeling reagents (d0-ABDMAP and d8-ABDMAP), (B) reaction of acylcarnitines with isotope labeling reagents, and (C) the workflow to identify acylcarnitines with isotope labeling method. reaction. Based on the chromatographic and mass spectrometric characteristics of the labeled products, specific identification of acylcarnitines was achieved. The schematic illustration for the profiling of acylcarnitines was shown in Figure 1. First of all, a pair of isotope labeling reagents, 1-(3-aminobutyl)-4-(dimethylamino)-pyridinium and its deuterated counterpart 1-(3-amino-d8-butyl)-4(dimethylamino)-pyridinium (d0-ABDMAP and d8ABDMAP) were synthesized (Figure 1A). The reagents contained primary amines which were reacted with carboxyl groups of acylcarnitines to form amide bonds. The reactive groups were connected to DMAP by the isotopically labeled moieties, i.e., d0-butyl and d8-butyl, to form positively charged reagents. The DMAP not only acted as ionizable groups, but also as conjugate groups to disperse the charges to stabilize the reagents. The synthetic isotope labeling reagents were characterized using MS, tandem mass spectrometry (MS/MS), 1H and 13C nuclear magnetic resonance spectra (shown in Figure S1, Supporting Information). The MS spectra showed that m/z of this pair of reagents differed by 8.0502 Da. This was the same as the difference between the d0-butyl and d8-butyl. Then, the labeling reagents were employed to selectively label carboxyl groups of acylcarnitines by condensation, generating doubly charged products (Figure 1B). The percentage of water in the sample greatly affected the reaction speed and efficiency (shown in Figure S2, Supporting Information). It meant that removing water from the samples was benefit for the reaction. Therefore, prior to labeling, samples were dried in order to achieve the maximum reaction rate and yield. The acylcarnitines could be almost completely labeled with the reagents under an anhydrous condition. Moreover, the reaction was carried out at room temperature without strong acid or alkali as catalyst. Compared to previously reported methods,9 the labeled products generated by our method were stable under such mild reaction conditions, avoiding the hydrolysis of the ester bond of acylcarnitines. The result was further confirmed by stability and recoveries evaluation (shown in Table S1, Supporting Information). The intra- and inter-day stability were smaller than 11.9%, and the recoveries were between 92.8% and 102.3%. Other validation
results also demonstrated that the method had good linearity and sensitivity. Therefore, our approach has shown a strong potential for the identification of acylcarnitines in biological samples. To profile urinary acylcarnitines, a pooled urine sample was divided into two equal portions and then reacted with d0ABDMAP and d8-ABDMAP, respectively (Figure 1C). Nontargeted profiling of the labeled samples was analyzed by ultrahigh performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS) with full scan mode. After data processing, d0- and d8-labeled acylcarnitines were screened by ShiftedIonsFinder, a software developed by Suzuki’s group,25 based on their chromatographic and mass spectrometric characteristics. The characteristics were obtained by analyzing d0- and d8labeled acylcarnitine standards. Because of their similar structures, the retention time of these two products was similar (Figure 2A). Due to the isotope effect caused by deuterium atoms, d8-labeled products were eluted a little earlier than d0labeled products. Therefore, their retention time were similar, but not the same. Their peak area ratio was 0.99, meaning a very close peak intensity. The isotopic peaks of d0-labeling (or d8labeling) product were differed by about 0.5 Da in MS spectra (Figure 2B). And the mass-to-charge ratio difference (Δm/z) between the d0- and d8-labeled acylcarnitines was 4.0260 Da (the theoretical value was 4.0251 Da), which was about half of the theoretical exact mass difference (Δm) of the labeling reagents (8.0502 Da). The results suggested that the labeled products were double charged peaks. That is because acylcarnitines contain one carboxyl group, they could only react with an equivalent amount of d0-ABDMAP (or d8-ABDMAP). Therefore, the Δm of the two products should be 8.0502 Da, the same as the difference of the labeling reagents. However, since the acylcarnitine itself contained a quaternary ammonium group, after reacting with a positively charged labeling reagent, the reaction product carried two positive charges (z = 2). Therefore, the Δm/z was 4.0251 Da, which was about half of Δm of the labeling reagents. The formation of the special Δm/z was associated with the distinctive molecular structure of acylcarnitines. Therefore, only compounds that contained both
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Analytical Chemistry
Figure 2. The chromatographic (A) and mass spectrometric characteristics of d0- and d8-labeled acylcarnitines (B). The difference of the two retention time was caused by isotope effect. one quaternary ammonium group and one carboxyl group in the molecule could generate such characteristic peaks. The mass spectral peaks of other metabolites were different from that of acylcarnitines. For metabolites without carboxyl groups, they could not react with the isotope labeling reagents, so that no paired peaks were formed. For monocarboxylic metabolites, they were capable of reacting with an equivalent amount of d0-ABDMAP or d8-ABDMAP. The Δm of the d0and d8-labeled products were 8.0502 Da and both of them had one positive charge (z = 1). Therefore the Δm/z of labeling monocarboxylic metabolites was 8.0502 Da (shown in Figure S3A, Supporting Information). For dicarboxylic metabolites, both of their carboxyl groups could react with the labeling reagents. So the Δm and z were 16.1004 Da and 2, respectively. As a result, the Δm/z of them still remained 8.0502 Da (shown in Figure S3B, Supporting Information). Similarly, for tricarboxylic metabolites, the Δm/z was also 8.0502 Da (shown in Figure S3C, Supporting Information). Only the difference among the three kinds of carboxyl-containing metabolites was that the isotopic peaks of each product were differed by 1/n Da, where n was the number of carboxyl groups. However, the number of carboxyl groups had no effect on the Δm/z of these d0- and d8-labeled carboxyl-containing metabolites. Taken together, all the Δm/z of these metabolites were 8.0502 Da, which was significantly different from that of acylcarnitines. The fragmentation pattern of known acylcarnitines provides important insight into the discovery and identification of unknown acylcarnitines. The d0- and d8-labeled C8:0carnitines had similar MS/MS spectra (Figures 3A and 3B). The common fragment ions of the two spectra were m/z 60, 86 and 123, which suggested that they did not contain any isotopic tags. In d0-labeled samples, fragment ions of m/z 116, 130, 177, 194 and 220 presented mass shifts to their corresponding ions (m/z 120, 134, 184, 202 and 227) in d8-labeled samples. The results indicated that these ions contained the whole or part of the isotopic tags. Based on these information, the possible fragmentation pathways were deduced, as shown in Figure 3C. The pair of characteristic fragment ions generated from the MS/MS spectra of d0- and d8-labeled acylcarnitines could be used for structure confirmation of unknown acylcarnitines. By searching the Human Metabolome Database (HMDB),26 we found that in addition to acylcarnitines, there were a small
number of metabolites possessing both a quaternary ammonium and carboxylic acid moieties as well, such as betaine, 4trimethylammoniobutanoic acid, and so on. These metabolites could also form chromatographic and mass spectra with three characteristics similar to acylcarnitines after they were reacted with the labeling reagents. However, their MS/MS spectra were different from those of acylcarnitines, such as the MS/MS spectra of labeled 4-trimethylammoniobutanoic acid shown in Figure S4, Supporting Information. Therefore, by combining the three features (a certain Δm/z, similar retention time, and carrying two positive charges) and the MS/MS spectra, these false positive signals can be largely excluded. It meant that the mass spectrometric characteristic of d0- and d8-labeled acylcarnitines may be used to distinguish the signals of acylcarnitines from that of other metabolites. In the previous reports,17-19 the identification of acylcarnitines was mainly carried out by virtue of their characteristic fragments (m/z 85.0284, 60.0808, 144.1019). The data obtained from high resolution MS provided a good basis for the identification of the structure. However, it should be noted that except for m/z 85.0284, the signal intensity of other fragment ions was very low. If the acylcarnitines were low abundance, these ions may not be detected. In such cases, the identification of acylcarnitines may be very difficult. And it may lead to false positive results. Because some other metabolites that were not acylcarnitines also had this fragment ion (such as the MS/MS spectrum of 3-hydroxy-C4-homoserine lactone, which could be found in MassBank database). In sharp contrast to the previous reports, we employed the characteristics of isotope labeling to identify candidate metabolites. Data from LC, MS, and MS/MS provided multiple lines of evidence for signal identification, which can greatly improve the accuracy of recognition. The acylcarnitines were discovered and identified by comparing the urine samples treated with d0-ABDMAP and d8ABDMAP, respectively. The total ions chromatograms of the d0- and d8-labeled products were similar (shown in Figure S5, Supporting Information). This is due to the light- and heavylabeled products had the similar retention behavior, forming nearly identical chromatograms. After data processing, the signals of labeled acylcarnitines were screened by using the software ShiftedIonsFinder based on their chromatographic and mass spectrometric characteristics as described above. The first cut was made to screen ion pairs that had the same retention time (± 0.2 min) and Δm/z of 4.0251 Da (± 15 ppm). A total of 332 paired peaks which met those criteria were selected from d0- and d8-labeled products. Secondly, by comparing their signal intensity, those peak pairs with intensity ratios in the range of 0.8-1.2 were retained. Finally, 247 pairs of doublecharged peaks were found to satisfy the characteristics of labeled acylcarnitines. Based on their m/z values, the exact molecular weight of the unlabeled acylcarnitines was obtained by subtracting the mass of the isotopic tags. The possible composition of the acyl group was inferred by elemental analysis. Then the MS/MS spectra of the d0- and d8-labeled acylcarnitines were analyzed to confirm the possible chemical structures. According to the above processing procedures, a total of 108 acylcarnitines were detected in human urine samples (Table S2, Supporting Information). Most of them were short- and medium-chain acylcarnitines, except for eleven long-chain acylcarnitines. This may be due to the fact that long-chain acylcarnitines were metabolized by cells to generate short- and
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medium-chain acylcarnitines. These metabolites were released into the urine, resulting in a large number of short- and mediumchain acylcarnitines. The result was consistent with the previously reported data.18 In addition, acylcarnitines with carbonyl groups or hydroxyl groups on the acyl chains, such as C4:0-OH and C6:0-CO (“OH” represents hydroxyl group, “CO” represents carbonyl group), were also detected by using the method. Although most of these identified acylcarnitines have no commercial standards, our proposed approach has the ability to confirm their structures. Compared to some previously reported methods, the number of acylcarnitines we detected was less. This was due to the difference in the instrument used and the scanning method. In this paper, we used full scan mode for data acquisition. It was a non-targeted method, which had lower sensitivity than that of targeted methods such as MRM or PRM. To combine the isotope labeling and MRM or PRM detection modes, more acylcarnitines may be detected. This is also the work we will carry out in the future. The special structure of acylcarnitines, i.e., having both a quaternary ammonium group and a carboxyl group, is the key to selective identification of acylcarnitines by isotope labeling strategy. Although positively charged isotope labeling reagents had been employed in previous studies to label carboxyl metabolites, they often utilized carboxyl metabolites rather than acylcarnitines to summarize the chromatographic and mass spectrometric characteristics of the labeled products.24,27,28 As a result, the corresponding specific characteristics were not suitable for the detection of acylcarnitines due to the formation of different Δm/z between the labeled products of acylcarnitines and carboxyl metabolites. Instead, in this work, we used
acylcarnitine standards to outline the characteristics of the labeled products and found out the special Δm/z of labeled acylcarnitines. Based on the characteristics, 108 acylcarnitines were discovered and identified. The results indicated that the proposed strategy developed in the work is highly selective and very valuable for the identification of acylcarnitines in biological samples. It also has potential application in quantitative method development for a large diversity of the metabolites, which may be great helpful for the studies of diseases associated with acylcarnitines metabolism.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental methods and materials, MS, MS/MS, 1H and 13C NMR spectra of isotope labeling reagents, optimization of reaction conditions, mass spectra of reaction products of other carboxylcontaining metabolites labeling with d0-ABDMAP and d8ABDMAP, MS and MS/MS spectra of d0- and d8-ABDMAP labeled 4-trimethylammoniobutanoic acid, and total ions chromatograms of urine samples labeled with d0-ABDMAP and d8-ABDMAP, validation of the isotope labeling method and detailed information of acylcarnitines detected in urine samples (PDF).
AUTHOR INFORMATION Corresponding Author * Email:
[email protected];
[email protected] ORCID Dan Gao: 0000-0003-1018-2656
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21675096).
REFERENCES
Figure 3. MS/MS spectra of (A) d0- and (B) d8-labeled C8:0carnitines and (C) the possible fragmentation pathways. *: molecular ion peak. **: double-charged peak. The values in brackets in (C): the corresponding fragment ions of d8-labeled C8:0-carnitine.
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