Abstract
Isotope labeling measurements using mass spectrometry can provide informative insights on the metabolic systems of various organisms. The detailed identification of carbon positions included in the fragment ions of dicarboxylic and tricarboxylic acids in central carbon metabolism is needed for precise interpretation of the metabolic states. In this study, fragment ions containing the carbon backbone cleavage of dicarboxylic and tricarboxylic in the Krebs cycle were investigated by using gas chromatography (GC)-electron ionization (EI)-MS and GC-EI-MS/MS. The positions of decarboxylation in the dicarboxylic and tricarboxylic acids were successfully identified by analyses using position-specific 13C-labeled standards prepared by in vitro enzymatic reactions. For example, carboxyl groups of C1 and C6 of trimethylsilyl (TMS)- and tert-butyldimethylsilyl (TBDMS)-derivatized malic and citric acids were primarily cleaved by EI. MS/MS analyses were also performed, and fragment ions of TBDMS-citric and α-ketoglutaric acids (αKG) with the loss of two carboxyl groups in collision-induced dissociation (CID) were observed.
Keywords: organic acids, fragmentation, GC-MS, electron ionization, collision-induced dissociation
INTRODUCTION
Isotope labeling experiments using mass spectrometry (MS) are one of the powerful methods to study metabolic systems. In particular, 13C-labeling of dicarboxylic and tricarboxylic acids in the Krebs cycle is measured in the fields of biotechnology, systems biology, and medical sciences.1–4) Since the interpretation of 13C-labeling data requires positional information about the carbon atoms in the fragment ions, identification of carbon atoms contained in the fragment ions as observed by MS and tandem-MS has been investigated.5–10) However, the carbon positions in the decarboxylated fragment ions derived from dicarboxylic and tricarboxylic acids have not been experimentally validated, resulting in the loss of rich information. In this study, the electron ionization (EI)- and collision-induced dissociation (CID)-fragmentation of the abundant dicarboxylic and tricarboxylic acids in the Krebs cycle, viz. citric, αKG, succinic, fumaric, and malic acids was investigated to maximize the accessible data from a single analysis. The organic acid-derived carbon atoms included in the fragment ions were successfully identified by the analyses of position-specific 13C-labeled standards synthesized by in vitro enzymatic reactions.
EXPERIMENTAL
Chemicals
Non-labeled standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). [1,2,3,4-13C]α-Ketoglutaric acid (99%), NaH13CO3 (99%), [1-13C]pyruvic acid (99%), [2-13C]pyruvic acid (99%), [3-13C]pyruvic acid (99%), and [1-13C]acetic acid (99%) were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Fully 13C-labeled organic acids were prepared from the extract of yeast cultured in a medium containing [U-13C]glucose as the sole carbon source following the previously described method.11)
Preparation of position-specific 13C-labeled standards
In vitro enzymatic reaction was used for the synthesis of position-specific 13C-labeled standards as described previously with minor modification.12) [4-13C]Malic acid was prepared via the reactions of phosphoenolpyruvate (PEP) carboxylase and malate dehydrogenase. Sixty-six millimolar Tris-HCl (pH=9), 10 mM PEP, 10 mM MgCl2, 20 mM NaH13CO3, 0.15 mM NADH, 4 units of PEP carboxylase from Zea mais leaves (Wako Pure Chemical Industries, Ltd. Corporation, Osaka, Japan), and 2 units of malate dehydrogenase from porcine heart (Wako Pure Chemical Industries, Ltd. Corporation) were gently mixed and incubated overnight at room temperature (Fig. S1a). [1-13C], [2-13C], and [3-13C]Malic acids were prepared via the sequential reactions of pyruvate kinase, PEP carboxylase, and malate dehydrogenase. Hundred millimolar Tris-HCl (pH=8), 10 mM ATP, 10 mM 13C-labeled pyruvic acid, 15 mM MgCl2, 20 mM NaHCO3, 10 mM NADH, 16 U of pyruvate kinase from rabbit muscle ca. 350 U/mg protein suspension (Wako Pure Chemical Industries, Ltd. Corporation), 4 U of PEP carboxylase from Zea mais leaves, and 2 U of malate dehydrogenase from porcine heart (Fig. S1a) were gently mixed and incubated overnight at room temperature. [1-13C], [2-13C], and [3-13C]Pyruvic acids were used for the synthesis of [1-13C], [2-13C], and [3-13C]malic acids, respectively. [1-13C], [5-13C], [6-13C], and [1,5-13C]Citric acids were prepared via the multiple reactions of pyruvate kinase, PEP carboxylase, acetyl-CoA synthetase (ACS), and citrate synthase (CS). Hundred millimolar Tris-HCl (pH=8), 10 mM pyruvic acid, 20 mM NaHCO3, 10 mM MgCl2, 10 mM ATP, 10 mM acetate, 10 mM CoA, 16 U of pyruvate kinase, 4 U of PEP carboxylase from Zea mais leaves, 2 U of citrate synthase, and 0.2 U of acetyl-CoA synthase were gently mixed and incubated overnight at room temperature. ACS and CS were obtained from the content of F-kit acetate (J.K. International, Tokyo, Japan) (Fig. S1b). Acetic acid, NaHCO3, and pyruvic acid were replaced with [1-13C]acetic acid, [1-13C]pyruvic acid, and NaH13CO3 for the synthesis of [1-13C], [5-13C], and [6-13C]citric acids, respectively.
Derivatization of organic acids
Standard solution of organic acids was evaporated to dryness by Speed Vac (Thermo Fischer Scientific, Waltham, MA, USA). The dried standards were derivatized by adding 50 μL of 40 mg/mL methoxyamine hydrochloride pyridine solution and incubated for 1 h at 30°C. Subsequently 50 μL of N-methyl-N-trimethylsilyltrifluoroacetamide containing 1% 2,2,2-trifluoro-N-methyl-N-(trimethylsilyl)-acetamide, chlorotrimethylsilane (Thermo Fischer Scientific) or N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide containing 1% tert-butyldimethylchlorosilane (Thermo Fischer Scientific) was added and the mixture was incubated for 1 h at 37 or 95°C for trimethylsilylation or tert-butyldimethylsilylation, respectively.8) After 1 h of cooling, an aliquot of the supernatant was subjected to the analysis.
GC-MS and GC-MS/MS analysis
GC-MS (GCMS-QP2010 Ultra, Shimadzu, Kyoto, Japan) and GC-MS/MS (GCMS-TQ8040, Shimadzu) equipped with DB-5MS+DG column ((30 m ×0.25 mm ID ×0.25 μm), Agilent Technologies, Santa Clara, CA, USA) were used.8) Analysis conditions were as follows: constant flow rate of helium at 1.0 mL/min; ion source temperature, 230°C; electron impact ionization, 70 eV; injection volume, 1 μL; injection, pulsed split (split ratio, 1 : 10); oven temperature, 60°C for 3.5 min, increased at a rate of 10°C/min to 325°C, and maintained at that temperature for 10 min or 70°C for 2 min, increased at a rate of 3°C/min to 280°C, and maintained at that temperature for 5 min for the analysis of TMS- and TBDMS-derivatives, respectively. Argon (200 kPa) was used as collision gas for the MS/MS analysis. Collision energy was optimized using Smart MRM (Shimadzu, Kyoto, Japan). The obtained raw MS and MS/MS spectra were deposited in MassBank.13)
RESULTS
EI-induced fragmentation of TMS- and TBDMS-derivatized organic acids
In this study, the EI- and CID-fragmentation of TMS- and TBDMS-derivatized dicarboxylic and tricarboxylic acids in the Krebs cycle was investigated by GC-EI-MS and GC-EI-MS/MS. To begin with, the TMS-derivatives of organic acids, one of the major analytes for metabolome analysis, were analyzed using GC-EI-MS. Figure 1 shows the EI-spectra of the TMS-derivatized malic acid. The ions with the highest intensity were 73 and 147, derived from TMS group.14) The other commonly observed fragment ions in TMS-derivatives were [M−15]+ and [M−117]+, in which the fragments were cleaved at the methyl group in TMS group and TMS-carboxyl group. The EI-spectra of TMS-derivatized malic acid produced intense [M−15]+ (m/z=335) and [M−117]+ (m/z=233) ions with relatively minor ions (m/z=101, 117, 189, 217, 265, and 307). These data are consistent with the previous study.14) To identify the carbon atoms included in these fragment ions, [U-13C]malic acid obtained from yeast cultured in [U-13C]glucose medium was derivatized and analyzed. As expected, the m/z of [M−15]+ and [M−117]+ ions shifted from 335 to 339, and from 223 to 226, respectively, demonstrating that [M−15]+ and [M−117]+ ions contained four and three carbons, respectively (Fig. 1b). Similarly, the fragment ions of [f101]+, [f117]+, [f189]+, [f217]+, [f265]+, and [f307]+ contained 2, 1, 2, 3, 1, and 3 carbons in a malic acid backbone (Fig. 1b). The cleaved TMS-carboxyl group in [M−117]+ could be C1 or C4 from a malic acid backbone. To determine this, position-specific 13C-labeled malic acid standard was prepared via in vitro sequential enzyme reactions of pyruvate kinase, phosphoenolpyruvate (PEP) carboxylase, and malate dehydrogenase supplemented with 13C pyruvic acid and 13C sodium bicarbonate (see experimental section, Fig. S1a). The analyses of TMS-derivatized [2-13C], [3-13C], and [4-13C]malic acids demonstrated the mass shift for [M−117]+ from 233 to 234 (Fig. 1d–f). However, no shift occurred in the spectrum of [1-13C]malic acid (Fig. 1c), indicating that the [M−117]+ loses the C1 in the malic acid backbone. Similarly, the following carbon positions in other fragment ions were identified: [f101]+ and [f189]+ had C2-3, [f117]+ had C4, [f265]+ had C2, and [f217]+ and [f307]+ had C2-3-4 from a malic acid backbone. The estimated structure of each fragment ions is illustrated in Fig. 2a. Although the structure of [f265]+ cannot be explained in a simple cleavage pattern, it is estimated that three TMS-O groups were possibly linked to the C2 of the malic acid backbone. The same approaches were applied for the survey of fragmentation in the TMS-derivatized citric, αKG, succinic, and fumaric acids (Supplementary materials 1). αKG was additionally methoxyaminated before the TMS derivatization. The fragment ions and the estimated structures of these TMS-derivatized organic acids with carbon backbone cleavage are summarized in Table 1 and Fig. 2b–e. The cleavage of TMS-carboxyl group in citric acid was successfully identified as C6 in the carbon backbone (Fig. 2b). The spectrum of αKG shows the generation of fragment ions with neither C1 nor C5 in the carbon backbone (Fig. 2c). The fragmentation of TMS-derivatized succinic and fumaric acids was estimated as Fig. 2d–e according to the structural symmetry.
Fig. 1. EI-induced fragmentation of TMS-derivatized malic acid.
(a) natural, (b) [U-13C], (c) [1-13C], (d) [2-13C], (e)[3-13C], and (f) [4-13C]malic acid.
Fig. 2. Estimated EI-fragmentation of TMS-derivatized organic acids.
(a) malic, (b) citric, (c) αKG, (d) succinic, and (e) fumaric acids. The position of demethylation in TMS group was not determined uniquely.
Table 1. Fragment ions with C–C bond cleavage of TMS- and TBDMS-derivatized organic acids by EI.
| Metabolites | m/z | Number of organic acid-derived carbons | Carbon skeleton | Estimated chemical formula | Estimated cleavage group |
|---|---|---|---|---|---|
| TMS derivatization | |||||
| Citric acid (4TMS) | 273 | 5 | C1-2-3-4-5 | C11H21O4Si2 | TMS-COO and TMS-OH |
| Citric acid (4TMS) | 347 | 5 | C1-2-3-4-5 | C13H27O5Si3 | TMS-COO and CH3 |
| Citric acid (4TMS) | 363 | 5 | C1-2-3-4-5 | C14H31O5Si3 | TMS-COO |
| αKG (1MEOX, 2TMS) | 112 | 4 | C1-2-3-4 | C5H6NO2 | TMS-COO and TMS-O |
| αKG (1MEOX, 2TMS) | 156 | 4 | C2-3-4-5 | C6H10NO2Si | TMS-COO, CH3 and CH3O |
| αKG (1MEOX, 2TMS) | 186 | 4 | C1-2-3-4 | C7H12NO3Si | TMS-COO and CH3 |
| Succinic acid (2TMS) | 116 | 2 | C1-2 or C3-4 | C4H8O2Si | TMS-COO-CH2 |
| Fumaric acid (2TMS) | 115 | 2 | C1-2 or C3-4 | C4H7O2Si | TMS-COO-CH |
| Fumaric acid (2TMS) | 143 | 3 | C1-2-3 or C2-3-4 | C6H11O2Si | TMS-COO |
| Malic acid (3TMS) | 101 | 2 | C2-3 | C4H9OSi | TMS-COO, TMS-COO and CH3 |
| Malic acid (3TMS) | 117 | 1 | C4 | C4H9O2Si | Except TMS-COO |
| Malic acid (3TMS) | 189 | 2 | C2-3 | C7H17O2Si2 | TMS-COO, CH3 and CO |
| Malic acid (3TMS) | 233 | 3 | C2-3-4 | C9H21O3Si2 | TMS-COO |
| Malic acid (3TMS) | 265 | 1 | C2 | C9H25O3Si3 | Unknown |
| Malic acid (3TMS) | 307 | 3 | C2-3-4 | C11H27O4Si3 | CO and CH3 |
| TBDMS derivatization | |||||
| Citric acid (4TBDMS) | 431 | 5 | C1-2-3-4-5 | C19H39O5Si3 | TBDMS-OH, TB and CO |
| Citric acid (4TBDMS) | 357 | 5 | C1-2-3-4-5 | C17H33O4Si2 | TBDMS-OH and TBDMS-CO |
| Citric acid (4TBDMS) | 299 | 5 | C1-2-3-4-5 | C13H23O4Si2 | TBDMS-OH, TBDMS-CO and TB |
| αKG (1MEOX, 2TBDMS) | 156 | 4 | C2-3-4-5 | C6H10NO2Si | TBDMS-CO, TB and CH3O |
| αKG (1MEOX, 2TBDMS) | 186 | 4 | C1-2-3-4 | C7H12NO3Si | TBDMS-COO and CH3 |
| Malic acid (3TBDMS) | 217 | 2 | C2-3 | C9H21O2Si2 | TBDMS-COO, CO, TB and CH3 |
| Malic acid (3TBDMS) | 317 | 3 | C2-3-4 | C15H33O3Si2 | TBDMS-CO |
| Malic acid (3TBDMS) | 349 | 1 | C2 | C15H37O3Si3 | Unknown |
| Malic acid (3TBDMS) | 391 | 3 | C2-3-4 | C17H39O4Si3 | TB and CO |
The fragmentation of TBDMS-derivatized organic acids was also explored in GC-EI-MS (Supplementary materials 2). The spectra show the relatively abundant [M−57]+, which contains all carbon backbones generated by the neutral loss of tert-butyl (TB) group. The backbone-derived carbon atoms included in decarboxylated fragment ions were determined by analyzing the synthesized position-specific 13C-labeled standards in a similar way (Table 1). The decarboxylated position of TBDMS organic acids was similar to that of TMS-derivatized ones. The examples include the cleavage of C6 in citric acid, C1 or C5 of αKG, and C1 of malic acid.
CID-induced fragmentation of TMS- and TBDMS-derivatized organic acids
Additionally, CID is a promising fragmentation mode in mass spectrometry. The CID-specific fragmentation was surveyed in TMS and TBDMS-derivatized organic acids using GC-EI-MS/MS (Supplementary materials 3 and 4). Fragment ions containing all carbon backbones, i.e., [M−57]+ and [M−15]+ for TBDMS- and TMS-derivatized organic acids, respectively, and relatively intense decarboxylated ions were chosen as precursor ions. The product ion scan of [f459]+ generated in the EI of TBDMS-derivatized citric acid, which contained all carbon backbone, produced the CID-specific ions such as [f253]+, [f327]+, and [f387]+ (Fig. 3a). The product ion scan of TBDMS-derivatized [U-13C]citric acid showed 6 mass shifts from 253 to 259 and from 327 to 333 and 4 mass shifts from 387 to 391 (Fig. 3b), indicating that [f387]+ contained 4 citric acid-derived carbon atoms while [f253]+ and [f327]+ contained all carbon atoms. The decarboxylated position of [f387]+ was investigated by the product ion scan of the various position-specific 13C-labeled citric acids. The m/z of product ions of TBDMS-derivatized [6-13C]citric acid remained at 387, demonstrating that carboxyl group of C6 was certainly lost in CID. Interestingly, the product ion scan of TBDMS-derivatized [1-13C] and [5-13C]citric acids produced ions with m/z of 387 and 388. These observations suggest that the carboxyl group of either C1 or C5 in TBDMS-citric acid was lost in CID. The assumption was validated by the presence of single ions of m/z=388 on the product ion spectra of [1,5-13C]citric acid. These data conclude that [f387]+ contained either C1-2-3-4 or C2-3-4-5. Similarly, the product ion scan of [f156]+ containing C1-2-3-4 of methoxyaminated and TBDMS-derivatized αKG generated in the EI produced [f112]+ with an additional decarboxylation in C1. Since these fragments were not observed in EI spectra, CID proved to be a complementary method to measure the 13C-labeling of decarboxylated fragment ions. The results are summarized in Table 2.
Fig. 3. CID-induced fragmentation of TBDMS-derivatized citric acid.
(a) natural, (b) [U-13C], (c) [1-13C], (d)[5-13C], (e)[6-13C] and (f)[1,5-13C]citric acid.
Table 2. Fragment ions with C–C bond cleavage of TMS- and TBDMS-derivatized organic acids by CID.
| Organic acids | Precursor ion | Product ion | |||||
|---|---|---|---|---|---|---|---|
| m/z | Carbon skeleton | m/z | Number of organic acid-derived carbons | Carbon skeleton | Estimated chemical formula | Estimated cleavage group from intact molecule | |
| TMS derivatization | |||||||
| Citric acid (4TMS) | 465 | C1-2-3-4-5-6 | 183 | 5 | C1-2-3-4-5 | C8H11O3Si | TMS-COO, TMS-OH and TMS-O |
| Citric acid (4TMS) | 465 | C1-2-3-4-5-6 | 257 | 5 | C1-2-3-4-5 | C10H17O4Si2 | CH3, TMS-COOH and TMS-OH |
| Citric acid (4TMS) | 465 | C1-2-3-4-5-6 | 347 | 5 | C1-2-3-4-5 | C13H27O5Si3 | CH3 and TMS-COOH |
| Citric acid (4TMS) | 465 | C1-2-3-4-5-6 | 273 | 5 | C1-2-3-4-5 | C11H21O4Si2 | TMS-OH and TMS-COOH |
| Citric acid (4TMS) | 363 | C1-2-3-4-5 | 183 | 5 | C1-2-3-4-5 | C8H11O3Si | TMS-COO, TMS-OH and TMS-O |
| Citric acid (4TMS) | 363 | C1-2-3-4-5 | 273 | 5 | C1-2-3-4-5 | C11H21O4Si2 | TMS-COO and TMS-OH |
| Citric acid (4TMS) | 273 | C1-2-3-4-5 | 183 | 5 | C1-2-3-4-5 | C8H11O3Si | TMS-COO, TMS-OH and TMS-O |
| αKG (1MEOX, 2TMS) | 288 | C1-2-3-4-5 | 244 | 4 | C1-2-3-4 | C10H22O2NSi2 | CH3O, CO, and CH3 |
| αKG (1MEOX, 2TMS) | 288 | C1-2-3-4-5 | 170 | 4 | C1-2-3-4 | C7H15NO2Si | CH3O and TMS-COOH |
| Fumaric acid (2TMS) | 245 | C1-2-3-4 | 217 | 3 | C1-2-3 or C2-3-4 | C8H17O3Si2 | CH3 and CO |
| Malic acid (3TMS) | 233 | C2-3-4 | 101 | 2 | C2-3 | C4H9OSi | TMS-COO, TMS-OOH and CH3 |
| Malic acid (3TMS) | 233 | C2-3-4 | 117 | 1 | C4 | C4H9O2Si | Except TMS-COO |
| Malic acid (3TMS) | 233 | C2-3-4 | 143 | 3 | C2-3-4 | C6H11O2Si | TMS-COO, TMS-OH |
| Malic acid (3TMS) | 233 | C2-3-4 | 189 | 2 | C2-3 | C8H21OSi2 | TMS-COO, CH3 and CO |
| Malic acid (3TMS) | 335 | C1-2-3-4 | 307 | 3 | C2-3-4 | C11H27O4Si3 | CH3 and CO |
| Malic acid (3TMS) | 335 | C1-2-3-4 | 263 | 2 | C2-3 | C10H27O2Si3 | CH3, CH2, CO and CO |
| Malic acid (3TMS) | 335 | C1-2-3-4 | 217 | 3 | C2-3-4 | C8H17O3Si2 | CH3 and TMS-COOH |
| Malic acid (3TMS) | 335 | C1-2-3-4 | 117 | 1 | C4 | C4H9O2Si | Except TMS-COO |
| TBDMS derivatization | |||||||
| Citric acid (4TBDMS) | 357 | C1-2-3-4-5 | 225 | 5 | C1-2-3-4-5 | C11H17O3Si | TBDMS-OH, TBDMS-COO, TBDMS-O |
| Citric acid (4TBDMS) | 357 | C1-2-3-4-5 | 313 | 4 | C1-2-3-4 or C2-3-4-5 | C16H33O2Si2 | TBDMS-OH, TBDMS-COO, CO and CH3 |
| Citric acid (4TBDMS) | 431 | C1-2-3-4-5 | 387 | 4 | C1-2-3-4 or C2-3-4-5 | C18H39O3Si3 | TBDMS-OH, TB, CO, CO and CH3 |
| Citric acid (4TBDMS) | 459 | C1-2-3-4-5-6 | 387 | 4 | C1-2-3-4 or C2-3-4-5 | C18H39O3Si3 | TBDMS-OH, TB, CO, CO and CH3 |
| αKG (1MEOX, 2TBDMS) | 346 | C1-2-3-4-5 | 186 | 4 | C1-2-3-4 | C7H12O3NSi | TB, and TBDMS-COO |
| αKG (1MEOX, 2TBDMS) | 346 | C1-2-3-4-5 | 156 | 4 | C1-2-3-4 | C6H10O2NSi | TB, TBDMS-COO and CH3O |
| αKG (1MEOX, 2TBDMS) | 156 | C1-2-3-4 | 112 | 3 | C2-3-4 | C5H10NSi | TB, TBDMS-COO CH3O, CO and CH3 |
| Malic acid (3TBDMS) | 419 | C1-2-3-4 | 217 | 3 | C2-3 | C9H21O2Si2 | TB, TBDMS-COO, CO and CH3 |
| Malic acid (3TDBMS) | 419 | C1-2-3-4 | 391 | 3 | C2-3-4 | C17H39O4Si3 | TB and CO |
| Malic acid (3TDBMS) | 419 | C1-2-3-4 | 403 | 4 | C1-2-3-4 | C17H39O4Si3 | TB and CH3 |
| Malic acid (3TDBMS) | 317 | C2-3-4 | 273 | 2 | C2-3 | C14H33OSi2 | TBDMS-COO, CO and CH3 |
DISCUSSION
In this study, the EI- and CID-fragmentation of TMS- and TBDMS-derivatized representative dicarboxylic and tricarboxylic acids in the Krebs cycle was explored. The analyses of the position-specific 13C-labeled standards prepared by in vitro enzymatic reactions successfully determined the position of cleaved carbon atom in the organic acid backbone (Tables 1, 2). The findings in our study are summarized as follows: (1) TMS-derivatized organic acids produced more fragment ions with C–C bond cleavage than TBDMS-derivatized ones by EI, (2) the carboxyl group next to hydroxylated carbon was primarily cleaved in EI, and (3) CID generated specific fragment ions with multiple decarboxylations.
As described in Fig. 1, various fragment ions were observed in TMS-derivatized malic acids by EI. In such case, it is possible to calculate the 13C-labeling of each carbon atom in organic acid backbone based on the 13C-labeling of several fragment ions with C–C bond cleavage.15) For example, 13C-labeling of C1 can be calculated computationally from the 13C-labeling of fragment ions with C1-2-3-4 and C2-3-4 of TMS-derivatized malic acid. Even the 13C-labeling of each carbon atom in TMS-derivatized malic acid backbone can be determined by measuring the 13C-labeling of novel fragment ions identified in this study, leading to maximization of the acquirable information from a single analysis. As compared to TMS-derivatization, TBDMS-derivatized organic acids produced relatively abundant ions with all carbon backbone ([M−57]+) and fewer fragment ions with C–C bond cleavage. For example, fragment ions containing C4 backbone appeared on the spectra of TMS- but not TBDMS-derivatized malic acid. These data demonstrate that TMS-derivatization is beneficial for generating EI-fragment ions with C–C bond cleavage.
Although the fragmentation patterns of TMS and TBDMS-derivatized organic acids were different, a common decarboxylation rule was found in EI. The analyses of the position-specific 13C-labeled standards validated that C1 of malic acid and C6 of citric acid was cleaved on EI-fragment ions. This suggests that the carboxyl group linked to the hydroxylated carbons in the organic acid backbone is primarily decarboxylated. The decarboxylated fragment ions of C1 and C5 in αKG were both found in EI, implying that the carboxyl group next to methoxyaminated carbon does not have a prior decarboxylation rule.
This study also highlights the fragmentation specificity of CID in derivatized organic acids as compared to EI. As shown in Table 2, CID can cleave two TBDMS-carboxyl groups. This difference may be explained by the fact that the radical cation could be generated and stabilized even in a single decarboxylation by EI, while the ions of univalent form could be kept stable during two parts of neutral loss of carboxyl group in CID. This insight sheds light on multiple CID events by ion-trap mass spectrometry for in-depth profiling of labeling information.
In this study, the EI- and CID-fragmentation of dicarboxylic and tricarboxylic acids was surveyed. The positions of organic acid-derived carbons contained in each fragment ion were successfully identified. These findings can contribute to the development of a fundamental theory of fragmentation in derivatized organic acids as well as the improvement of 13C-labeling experiments for biological system.
Acknowledgments
We thank Prof. Eiichiro Fukusaki (Osaka University, Japan) for his helpful comments. This research was partially supported by Grants-in-Aid for Scientific Research on Innovative Areas (17H06303) and Grant-in-Aid for JSPS Fellows (15J05988).
Mass Spectrom (Tokyo) 2019; 8(1): A0073
Supplementary Data
References
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