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Characterization of phthiocerol and phthiodiolone dimycocerosate esters of M. tuberculosis by multiple-stage linear ion-trap MS

Open AccessPublished:November 16, 2015DOI:https://doi.org/10.1194/jlr.D063735
      Both phthiocerol/phthiodiolone dimycocerosate (PDIM) and phenolic glycolipids are abundant virulent lipids in the cell wall of various pathogenic mycobacteria, which can synthesize a wide range of complex high-molecular-mass lipids. In this article, we describe linear ion-trap MSn mass spectrometric approach for structural study of PDIMs, which were desorbed as the [M + Li]+ and [M + NH4]+ ions by ESI. We also applied charge-switch strategy to convert the mycocerosic acid substituents to their N-(4-aminomethylphenyl) pyridinium (AMPP) derivatives and analyzed them as M + ions, following alkaline hydrolysis of the PDIM to release mycocerosic acids. The structural information from MSn on the [M + Li]+ and [M + NH4]+ molecular species and on the M + ions of the mycocerosic acid-AMPP derivative affords realization of the complex structures of PDIMs in Mycobacterium tuberculosis biofilm, differentiation of phthiocerol and phthiodiolone lipid families and complete structure identification, including the phthiocerol and phthiodiolone backbones, and the mycocerosic acid substituents, including the locations of their multiple methyl side chains, can be achieved.
      Both phthiocerol/phthiodiolone dimycocerosate (PDIM) esters and phenolic glycolipids (PGLs) are dimycocerosate esters (DIMs) produced by pathogenic mycobacteria. PDIMs were originally isolated from Mycobacterium tuberculosis (
      • Asselineau J.
      Sur quelques substances ii 60 atomes de carbone isolees des lipides de souches humaines de Mycobacterium tuberculosis.
      ,
      • Noll H.
      The chemistry of some native constituents of the purified wax of Mycobacterium tuberculosis.
      ,
      • Reeves R.E.
      • Anderson R.J.
      The chemistry of the lipids of tubercle bacilli: XLVIII. The occurrence of phthiocerol in the wax from various strains of the human tubercle bacillus.
      ,
      • Stodola F.H.
      • Anderson R.
      The chemistry of the lipids of tubercle bacilli: XLVI. Phthiocerol, a new alcohol from the wax of the human tubercle bacillus.
      ) and are specific tuberculosis biomarkers (
      • Redman J.E.
      • Shaw M.J.
      • Mallet A.I.
      • Santos A.L.
      • Roberts C.A.
      • Gernaey A.M.
      • Minnikin D.E.
      Mycocerosic acid biomarkers for the diagnosis of tuberculosis in the Coimbra Skeletal Collection.
      ,
      • Minnikin D.E.
      • Kremer L.
      • Dover L.G.
      • Besra G.S.
      The methyl-branched fortifications of Mycobacterium tuberculosis.
      ). They are among the most abundant lipids in the cell wall of various pathogenic mycobacteria, including M. tuberculosis, M. bovis, M. leprae, M. kansasii, M. microti, and M. marinum (
      • Onwueme K.C.
      • Vos C.J.
      • Zurita J.
      • Ferreras J.A.
      • Quadri L.E.N.
      The dimycocerosate ester polyketide virulence factors of mycobacteria.
      ,
      • Yu J.
      • Tran V.
      • Li M.
      • Huang X.
      • Niu C.
      • Wang D.
      • Zhu J.
      • Wang J.
      • Gao Q.
      • Liu J.
      Both phthiocerol dimycocerosates and phenolic glycolipids are required for virulence of Mycobacterium marinum.
      ,
      • Daffé M.
      • Laneelle M.A.
      Distribution of phthiocerol diester, phenolic mycosides and related compounds in mycobacteria.
      ,
      • Minnikin D.E.
      • Dobson G.
      • Goodfellow M.
      • Magnusson M.
      • Ridella M.
      Distribution of some mycobacterial waxes based on the phthiocerol family.
      ), which are known to synthesize a range of complex high-molecular-mass lipids (
      • Onwueme K.C.
      • Vos C.J.
      • Zurita J.
      • Ferreras J.A.
      • Quadri L.E.N.
      The dimycocerosate ester polyketide virulence factors of mycobacteria.
      ). PGLs are produced by the same set of pathogenic mycobacteria species, except that in M. tuberculosis only a subset of clinical isolates belonging to the W-Beijing family (
      • Reed M.B.
      • Domenech P.
      • Manca C.
      • Su H.
      • Barczak A.K.
      • Kreiswirth B.N.
      • Kaplan G.
      • Barry C.E.
      A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response.
      ) produces PGLs.
      Phthiocerol and phenolphthiocerol and other lipids such as mycolic acids and methyl-branched FAs in cell wall are among those that have been most extensively studied in terms of their biosynthesis and the role in M. tuberculosis virulence in vivo (
      • Kolattukudy P.E.
      • Fernandes N.D.
      • Azad A.K.
      • Fitzmaurice A.M.
      • Sirakova T.D.
      Biochemistry and molecular genetics of cell-wall lipid biosynthesis in mycobacteria.
      ). In pathogenesis, the role of PDIMs of M. tuberculosis was recognized by the studies that identified that mutants of M. tuberculosis were unable to either produce or properly localize PDIMs to the cell envelopeand that demonstrated that PDIM-deficient strains were attenuated in animal models of infection (
      • Yu J.
      • Tran V.
      • Li M.
      • Huang X.
      • Niu C.
      • Wang D.
      • Zhu J.
      • Wang J.
      • Gao Q.
      • Liu J.
      Both phthiocerol dimycocerosates and phenolic glycolipids are required for virulence of Mycobacterium marinum.
      ,
      • Reed M.B.
      • Domenech P.
      • Manca C.
      • Su H.
      • Barczak A.K.
      • Kreiswirth B.N.
      • Kaplan G.
      • Barry C.E.
      A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response.
      ,
      • Rousseau C.
      • Winter N.
      • Pivert E.
      • Bordat Y.
      • Neyrolles O.
      • Avé P.
      • Huerre M.
      • Gicquel B.
      • Jackson M.
      Production of phthiocerol dimycocerosates protects Mycobacterium tuberculosis from the cidal activity of reactive nitrogen intermediates produced by macrophages and modulates the early immune response to infection.
      ,
      • Cox J.S.
      • Chen B.
      • McNeil M.
      • Jacobs W.R.
      Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice.
      ,
      • Camacho L.R.
      • Ensergueix D.
      • Perez E.
      • Gicquel B.
      • Guilhot C.
      Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis.
      ,
      • Camacho L.R.
      • Constant P.
      • Raynaud C.
      • Lanéelle M-A.
      • Triccas J.A.
      • Gicquel B.
      • Daffé M.
      • Guilhot C.
      Analysis of the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis: evidence that this lipid is involved in the cell wall permeability barrier.
      ,
      • Murry J.P.
      • Pandey A.K.
      • Sassetti C.M.
      • Rubin E.J.
      Phthiocerol dimycocerosate transport is required for resisting interferon-γ–independent immunity.
      ). Recently, M. tuberculosis and its close pathogenic relative M. marinum were reported to manipulate macrophage recruitment through coordinated use of membrane PDIM and PGLs to initiate infections (
      • Cambier C.J.
      • Takaki K.K.
      • Larson R.P.
      • Hernandez R.E.
      • Tobin D.M.
      • Urdahl K.B.
      • Cosma C.L.
      • Ramakrishnan L.
      Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids.
      ). However, the precise role of these molecules in the course of infection remains largely unknown, and their role in the multiplication of mycobacteria from the tuberculosis complex in organs other than the lungs is also unclear.
      PDIM (Fig. 1) and PGL respectively consist of a long-chain 3-methoxy, 4-methyl, 9,11-dihydroxy glycol (phthiocerol) and a p-glycosylated phenylglycol (glycosyl phenolphthiocerol) backbone diesterified with di-, tri-, and tetra-methyl-branched long-chain mycocerosic (mycoceranic) acids (
      • Minnikin D.E.
      • Dobson G.
      • Goodfellow M.
      • Magnusson M.
      • Ridella M.
      Distribution of some mycobacterial waxes based on the phthiocerol family.
      ,
      • Goren M.B.
      • Brennan P.J.
      Mycobacterial lipids: chemistry and biologic activities. In Tuberculosis.
      ,
      • Minnikin D.E.
      • Goodfellow M.
      Lipid Com­position in the Classification and Identification of Acid-Fast Bacteria.
      ). Dependent on the species, the long-chain diol backbone ranges in size from C25 to C36, and the mycocerosic acid chain ranges from C23 or C24 to C32 (
      • Reeves R.E.
      • Anderson R.J.
      The chemistry of the lipids of tubercle bacilli: XLVIII. The occurrence of phthiocerol in the wax from various strains of the human tubercle bacillus.
      ,
      • Stodola F.H.
      • Anderson R.
      The chemistry of the lipids of tubercle bacilli: XLVI. Phthiocerol, a new alcohol from the wax of the human tubercle bacillus.
      ,
      • Minnikin D.E.
      • Dobson G.
      • Goodfellow M.
      • Magnusson M.
      • Ridella M.
      Distribution of some mycobacterial waxes based on the phthiocerol family.
      ,
      • Smith D.W.
      • Randall H.M.
      • Gastambide-Odier M.M.
      • Koevoet A.L.
      The characterization of mycobacterial strains by the composition of their lipide extracts.
      ,
      • Demartreau-Ginsburg H.
      • Lederer E.
      • Ryhage R.
      • Stallberg-Stenhagen S.
      • Stenhagen E.
      Structure of phthiocerol.
      ). Other variants with 3-keto or 3-hydroxy diols also exist. The phthiocerol family consists of phthiocerol A, phthiocerol B, phthiodiolone A, and phthiotriol A, of which phthiocerol A and phthiodiolone A are the most commonly present. In M. marinum, a major analogous PGL family consisting of the long-chain β-diol backbone modified with a phenolic group at the terminal, to which a 3-O-methylrhamnose is β-O-linked to the phenol ring, is also present (
      • Daffé M.
      • Laneelle M.A.
      Distribution of phthiocerol diester, phenolic mycosides and related compounds in mycobacteria.
      ,
      • Alibaud L.
      • Rombouts Y.
      • Trivelli X.
      • Burguière A.
      • Cirillo S.L.G.
      • Cirillo J.D.
      • Dubremetz J-F.
      • Guérardel Y.
      • Lutfalla G.
      • Kremer L.
      A Mycobacterium marinum TesA mutant defective for major cell wall-associated lipids is highly attenuated in Dictyostelium discoideum and zebrafish embryos.
      ).
      Figure thumbnail gr1
      Fig. 1Structure of PDIM ester observed in this study.
      MS has played important roles in the elucidation of the structures of PDIMs and PGLs. For example, Minnikin et al. (
      • Minnikin D.E.
      • Dobson G.
      • Goodfellow M.
      • Magnusson M.
      • Ridella M.
      Distribution of some mycobacterial waxes based on the phthiocerol family.
      ,
      • Minnikin D.E.
      • Dobson G.
      • Hutchinson I.G.
      Charac­terization of phthiocerol dimycocerosates from Mycobacterium tuberculosis.
      ,
      • Dobson G.
      • Minnikin D.E.
      • Besra G.S.
      • Mallet A.I.
      • Magnusson M.
      Characterisation of phenolic glycolipids from Mycobacterium marinum.
      ,
      • Nicoara S.C.
      • Minnikin D.E.
      • Lee O.C.Y.
      • O'Sullivan D.M.
      • McNerney R.
      • Pillinger C.T.
      • Wright I.P.
      • Morgan G.H.
      Development and optimization of a gas chromatography/mass spectrometry method for the analysis of thermochemolytic degradation products of phthiocerol dimycocerosate waxes found in Mycobacterium tuberculosis.
      ) and Daffe et al. (
      • Daffé M.
      • Laneelle M.A.
      Distribution of phthiocerol diester, phenolic mycosides and related compounds in mycobacteria.
      ,
      • Daffé M.
      • Draper P.
      The envelope layers of mycobacteria with reference to their pathogenicity In Advances in Microbial Physiology.
      ,
      • Daffé M.
      • Lacave C.
      • Lanéelle M.-A.
      • Lanéelle G.
      Structure of the major triglycosyl phenol-phthiocerol of Mycobacterium tuberculosis (strain Canetti).
      ,
      • Constant P.
      • Perez E.
      • Malaga W.
      • Lanéelle M-A.
      • Saurel O.
      • Daffé M.
      • Guilhot C.
      Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex: evidence that all strains synthesize glycosylated p-hydroxybenzoic methyl esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene.
      ) used GC/MS analysis together with NMR spectroscopy for complete characterization of DIMs, following extraction steps, chromato­graphic separations, and chemical reactions. Recently, MALDI-TOF (
      • Camacho L.R.
      • Constant P.
      • Raynaud C.
      • Lanéelle M-A.
      • Triccas J.A.
      • Gicquel B.
      • Daffé M.
      • Guilhot C.
      Analysis of the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis: evidence that this lipid is involved in the cell wall permeability barrier.
      ,
      • Constant P.
      • Perez E.
      • Malaga W.
      • Lanéelle M-A.
      • Saurel O.
      • Daffé M.
      • Guilhot C.
      Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex: evidence that all strains synthesize glycosylated p-hydroxybenzoic methyl esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene.
      ,
      • Siméone R.
      • Constant P.
      • Guilhot C.
      • Daffé M.
      • Chalut C.
      Identification of the missing trans-acting enoyl reductase required for phthiocerol dimycocerosate and phenolglycolipid biosynthesis in Mycobacterium tuberculosis.
      ) and ESI-Fourier transform ion cyclotron resonance (
      • Jain M.
      • Petzold C.J.
      • Schelle M.W.
      • Leavell M.D.
      • Mougous J.D.
      • Bertozzi C.R.
      • Leary J.A.
      • Cox J.S.
      Lipidomics reveals control of Mycobacterium tuberculosis virulence lipids via metabolic coupling.
      ) MS have also been applied for profiling PDIM and PGLs. However, the ESI/MS/MS method useful for direct structural identification of these complex lipids has not been established. Here, we describe MSn linear ion trap (LIT) with high-resolution MS toward characterization of the structures of PDIMs, which were desorbed as the [M + Alk]+ (Alk = Li, Na, NH4) ions by ESI. This mass spectrometric approach affords realization of the structures of this lipid family isolated from the biofilm formed by M. tuberculosis, including the identities of the mycocerosic acid side chains and the phthiocerol backbone.

      MATERIALS AND METHODS

      Chemicals

      All chemicals and solvents were purchased from Fisher Scientific (Waltham, MA). Standard C30/C30-mC26:0 PDIM (refer to Nomenclature) was synthesized as previously described (
      • Casas-Arce E.
      • ter Horst B.
      • Feringa B.L.
      • Minnaard A.J.
      Asymmetric total synthesis of PDIM A: a virulence factor of Mycobacterium tuberculosis.
      ).

      Bacterial strains and growth conditions

      M. tuberculosis Erdman was cultured at 37°C in Middlebrook 7H9 or Middlebrook 7H10 agar plates supplemented with 60 μl/l oleic acid, 5 g/l BSA, 2 g/l dextrose, 0.003 g/l catalase, 0.5% glycerol, and 0.05% Tween 80 (broth) or in Sauton's liquid medium unless otherwise indicated. Bacterial biofilms were inoculated with stationary phase planktonic cultures into Sauton's media at a 1:100 dilution. Culture vessels were closed tightly to restrict oxygen for 3 weeks and then vented, as described by Ojha (
      • Ojha A.K.
      • Baughn A.D.
      • Sambandan D.
      • Hsu T.
      • Trivelli X.
      • Guerardel Y.
      • Alahari A.
      • Kremer L.
      • Jacobs W.R.
      • Hatfull G.F.
      Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria.
      ).

      Bacterial lipid extraction

      Bacterial biofilms were harvested at the indicated time, pelleted, and boiled for 30 min. Samples were then extracted twice by adding chloroform-methanol (2:1), sonicating for 5 min, incubating 1 h, and centrifuging, and the organic phase from two extractions (twice) was pooled and dried under nitrogen gas. Lipids were dissolved in chloroform-methanol (2:1) before analysis.

      LC/MS fractionation of PDIMs

      Preparative HPLC experiments were carried out using a Thermo Scientific (San Jose, CA) TSQ Vantage mass spectrometer with Thermo Accela UPLC operated by Xcalibur software. Separation of lipid was achieved by a Supelco 100 × 2.1 mm (2.7 μ particle size) Ascentis C-8 column at a flow rate of 260 μl/min. The mobile phase contained 10 mM ammonium formate (pH 5.0) in solvent A-acetonitrile-water (60:40, v/v), solvent B-2-propanol-acetonitrile (90:10, v/v); a gradient elution in the following manner was applied: 68% A, 0–1.5 min; 68–55% A, 1.5–4 min; 55–48% A, 4–5 min; 48–42% A, 5–8 min; 42–34% A, 8–11 min; 34–30% A, 11–14 min; 30–25% A, 14–18 min; 25–3% A, 18–23 min; 3–0% A, 25–30 min; 0% A, 30–35 min; 68% A, 35–40 min. During fractionation, ∼95% of the lipid was sent to a fraction collector, and a small percentage (5%) of the lipid was sent to the mass spectrometer via a tee to identify the structure. The PDIM fraction was eluted at 30.5–34.5 min, and family of phthiocerol dimycocerosate eluted earlier than that of phthiodiolone dimycocerosate (see supplementary Fig. 1B). PDIM fractions from several injections were collected and pooled, dried under a stream of nitrogen, and subjected to further structural analyses as described subsequently.

      Alkaline hydrolysis and preparation of N-(4-aminomethylphenyl) pyridiniumderivative with reagent

      Alkaline hydrolysis to yield myroceranoic acids from PDIM was carried out following the protocol as previously described (
      • Tatituri R.V.
      • Wolf B.
      • Brenner M.
      • Turk J.
      • Hsu F-F.
      Characterization of polar lipids of Listeria monocytogenes by HCD and low-energy CAD linear ion-trap mass spectrometry with electrospray ionization.
      ) with modification. To the tube containing dried PDIM, 400 μl methanol, 200 μl diethylether, and 200 μl tetrabutylammonium hydroxide (40 wt% solution in water) were added. The tube was capped and heated at 100°C overnight and cooled to room temperature, and 50 μl HCl (37%) was added. Following addition of 1 ml water and 1 ml chloroform, vortexing for 1 min, and centrifuging at 1,200 gfor 2 min, the organic layer was transferred to another tube and was washed twice with 1 ml water. The final organic layer containing FFAs was dried under nitrogen, and N-(4-aminomethylphenyl) pyridinium (AMPP) derivative was made with the AMP+ Mass Spectrometry Kit, according to the manufacturer's instruction. Briefly, the dried sample was resuspended in 20 μl ice-cold acetonitrile/N,N-dimethylformamide (4:1, v/v), and 20 μl of ice-cold 1 M 3-(dimethylaminopropyl)ethyl carbodiimide hydrochloride in water was added. The vial was briefly mixed on a vortex mixer and placed on ice. To the sample tube, 10 μl of 5 mM N-hydroxybenzotriazole and 30 μl of 15 mM AMPP (in distilled acetonitrile) were added. After the solution was heated at 65°C for 30 min and cooled to room temperature, 1 ml of water and 1 ml of n-butanol were added. The final solution containing FA-AMPP derivative was vortexed for 1 min and centrifuged for 3 min at 1,200 g. The organic layer was transferred to another vial, dried under a stream of nitrogen, and stored at −20°C until use.

      MS

      Both high-resolution (R = 100,000 at m/z 400) higher energy collision activation dissociation and low-energy collision-induced dissociation (CID) MS/MS experiments were conducted on a Thermo Scientific LTQ Orbitrap Velos mass spectrometer with Xcalibur operating system. Lipid extracts in chloroform-methanol (2:1) were infused (1.5 μl/min) to the ESI source, where the skimmer was set at ground potential, the electrospray needle was set at 4.0 kV, and temperature of the heated capillary was 300°C. The automatic gain control of the ion trap was set to 5 × 104, with a maximum injection time of 50 ms. Helium was used as the buffer and collision gas at a pressure of 1 × 10−3 mbar (0.75 mTorr). The MSn experiments were carried out with an optimized relative collision energy ranging from 30% to 45%, an activation q value at 0.25, and the activation time at 10 ms to leave a minimal residual abundance of precursor ion (∼20%). The mass selection window for the precursor ions was set at 1 Da wide to admit the monoisotopic ion to the ion trap for CID for unit resolution detection in the ion trap or high-resolution accurate mass detection in the Orbitrap mass analyzer. Mass spectra were accumulated in the profile mode, typically for 2–10 min for MSn spectra (n = 2,3,4).

      Nomenclature

      For simplicity, the terms phthiocerol dimycocerosate and phthiodiolone dimycocerosate, abbreviated as PDIM, will be used for all the families of the parent waxes without implying one particular stereochemistry for the component of the multimethyl-branched acids. The term phthioglycol, a modification of Stendal's original nomenclature (
      • Stendal N.
      The presence of a glycol in the wax of tubercle bacilli.
      ), will be used to refer to the family of compounds, and the term phthiocerol will be reserved for the original 3-methoxy congener. Thus, the abbreviation of the phthiocerol backbone of a PDIM, for example, the 3-methoxy, 4-methyl, 9,11-dihydroxy hexaeicosane, is designated as mC27:0 to reflect the C27 chain length with a methoxy group attached at C-3; glycol backbones with 3-keto, 4-methyl 9,11-dihydroxy hexaeicosane and with 3-hydroxy, 4-methyl 9,11-dihydroxy hexaeicosane are designated as kC27:0 and as hC27:0, respectively. The two mycocerosate residues consisting of two tetramethyl-branched C30-acyl chains located at 9 and 11, for example, are abbreviated as C30/C30. Therefore, a phthioglycol with a respective mC27:0 and kC27:0 backbone and two C30-acyl chains is designated as C30/C30-mC27:0 PDIM and C30/C30-kC27:0-PDIM, respectively.

      RESULTS

      CID MSn on the 3-methoxy, 4-methyl, 9,11-di-(3,5,7,9-tetramethyloctaeicosanoyl) tetratriacontanediol (C32:0/C32:0-mC35:0 PDIM) standard

      PDIMs readily form [M + Alk]+ ions (Alk = Li, Na, NH4) when subjected to ESI in the presence of alkaline ion in positive-ion mode. Corresponding ions in the fashion of [M + X] (X = Cl, HCO2) were also observed in the negative-ion mode; however, CID MSn on these latter ions failed to provide sufficient information for structural identification and will not be discussed further.
      To gain insight into the fragmentation processes, we first performed MSn on the [M + Li]+ and [M + Na]+ adduct ions of C32:0/C32:0-mC35:0 PDIM standard to explore their utilities toward structural determination of PDIMs. As shown in Fig. 2A, the MS2 spectrum of the [M + Li]+ ion at m/z 1,486.5 contained ions at m/z 1,006.0 arising from loss of 3,5,7,9-tetramethyloctaeicosanoic acid residue (32:0) and at m/z 525 (1,006.0–480.5) arising from further loss of the remaining 32:0-FA substituent (480.5 Da), along with ion at m/z 487, representing a lithiated 32:0-FA cation. The consecutive loss of the FA substituent was further supported by the MS3 spectrum of the ions of m/z 1,006 (1,486 → 1,006; Fig. 2C), which contained major ions at m/z 525 and 487. Similarly, the MS2 spectrum of the corresponding [M+ Na]+ ions at m/z 1,502.5 (Fig. 2B) contained ions at m/z 1,022.1, 541, and 503 that are 16 Da heavier, and the MS3 spectrum of the ions of m/z 1,022 (1,502 → 1,022; data not shown) also contains the ions of m/z 541 and 503, which are also 16 Da heavier, consistent with the losses of the 32:0-FA moieties.
      Figure thumbnail gr2
      Fig. 2The MS2 spectra of the [M + Li]+ ion of C32:0/C32:0-mC34:0 PDIM at m/z 1,486.5 (A), of the corresponding [M + Na]+ ions at m/z 1,502.5 (B), and the consecutive MS3 spectrum of the ion of m/z 1,006.1 (1,486.5 → 1,006.1) (C); the MS4 spectrum of the ion of 525 (1,486.5 → 1,006.1→ 525) (E) from the [M + Li]+ ion. Panel D shows the MS2 spectrum of the corresponding [M + NH4]+ ion at m/z 1,497.5, and the MS3 spectrum of the ion of m/z 1,000.1 (1,497.5 → 1,000.1) (F), MS5 spectrum of the ion of m/z 487 (1,497.5 → 1,000.1 → 519 → 487) (G), MS6 spectrum of the ions of m/z 417 (1,497.5 → 1,000.1 → 521 → 487 → 417) (H) support the fragmentation processes depicted in . Please also note that the mass discrepancy of the labeling in all the figures is due to mass defect.
      In contrast, the MS2 spectrum of the [M+ NH4]+ ion at m/z 1,497.5 (Fig. 2D) is dominated by the ion at m/z 1,000 arising from losses of the 3,5,7,9-tetramethyloctaeicosanoic acid and NH3, similar to that observed for the [M+ NH4]+ ion of triacylglycerol (TAG) (
      • Hsu F-F.
      • Turk J.
      Electrospray ionization multiple-stage linear ion-trap mass spectrometry for structural elucidation of triacylglycerols: assignment of fatty acyl groups on the glycerol backbone and location of double bonds.
      ). The spectrum also contained the ion at m/z 968, arising from further loss of CH3OH, along with the ions at m/z 519 from additional loss of the 32:0-FA substituent, and at m/z 487 (519 – CH3OH) arising from further loss of the methoxy group. The losses of the FA substituents and NH3, and methanol were supported by high-resolution MS (data not shown). The observation of the ions at m/z 968 (1,000 – CH3OH) and at 487 (519 – CH3OH) corresponding to loss of CH3OH residue, supports the notion that the phthiocerol possesses a methoxy side chain, and the molecule belongs to the 3-methoxy 4-methyl PDIM family.
      It appeared that the 32:0-FA substituent at C11 was first cleaved from the phthiocerol backbone via a charge-remote fragmentation process to yield a lithiated ion of m/z 1,006.1 (Fig. 2A), which possesses a double bond at C-11 (Scheme 1). This is followed by elimination of the remaining 32:0-FA at C9 to form the lithiated ion of m/z 525 that consists of a conjugated double bond at C9 and C11. This assumption is based on the findings that the MS4 spectrum of the ion of m/z 525 (1,486 → 1,006→ 525; Fig. 2E) contained the major ion at m/z 493, arising from loss of the methoxy group (loss as CH3OH) attached to the phthiocerol chain, along with ions at m/z 231 arising from allylic cleavage of the C13-C14 bond and at m/z 161 arising from cleavages that may eliminate an alkene, acetylene, and H2 (Scheme 1).
      Similarly, the 32:0-FA residue at C11 was also preferentially cleaved (Fig. 2D), resulting in the elimination of NH3 and 32:0-FA residues to form the ion of m/z 1,000 in which the proton may reattach to the carboxylate group of the remaining 32:0-FA (Scheme 2). Further dissociation of the ion of m/z 1,000 (1,497 → 1,000; Fig. 2F) eliminates the 32:0-FA substituent at C9 to give rise to the major ion of m/z 519, which may represent a protonated phthiocerol ion possessing a conjugated double bond with proton relocated at the methoxy side chain. This is followed by loss of a methanol molecule to yield the ion of m/z 487, in which the charge site may situate at C7 via hydrogen shift. These fragmentation processes leading to the ions of m/z 487 were supported by the MS4 spectrum of the ion of m/z 519 (1,497 → 1,000 → 519; data not shown). The loss of the 32:0-FA substituent is also consistent with the observation of the ion at m/z 481, representing a protonated 32:0-FA ion, in Fig. 2F.
      The dissociation of the ion of m/z 487 (1,497 → 1,000 → 521 → 487; Fig. 2G) gave rise to the ion series of m/z 445, 431, 417, 403, 389, 375, and so forth, along with the ion series of m/z 151, 165, 179, 193, 207, 221, and so forth (intensity in the descending order), arising from cleavages of the C-C bonds of phthiocerol chain. The observation of these two ion series is consistent with the notion that the charge may primarily reside at C7, and cleavages of these C-C bonds may be similar to the “mobile proton” model (
      • Wysocki V.H.
      • Tsaprailis G.
      • Smith L.L.
      • Breci L.A.
      Mobile and localized protons: a framework for understanding peptide dissociation.
      ,
      • Paizs B.
      • Suhai S.
      Fragmentation pathways of protonated peptides.
      ), in which more than one charge sites are energetically and/or kinetically favored due to proton rearrangement along the phthiocerol backbone. For example, C-C bond cleavages distal to the methyl side chain terminal from precursor ions possessing charge located at C7 gave rise to ions at m/z 193, 207, 221, 235, and so forth; while ions at m/z 445, 431, 417, 403, 389, and so forth arose from C-C cleavages from the methyl side chain terminal. Further dissociations of the ions of m/z 431, 417, and 403 also gave rise to ions of m/z 389, 375, and 361 by loss of propylene residue (
      • Hsu F.F.
      • Turk J.
      Elucidation of the double-bond position of long-chain unsaturated fatty acids by multiple-stage linear ion-trap mass spectrometry with electrospray ionization.
      ); while ions of m/z 151 (loss of C19-alkene), 137 (loss of C20-alkene), and 123 (loss of 21:0-alkene) can also arise from, for example, m/z 417 by consecutive fragmentation processes that eliminate an alkene residue (Scheme 2). These fragmentation processes are supported by MS6 spectra of the ions of m/z 431 (1,497 → 1,000 → 521 → 487 → 431; not shown), 417 (1,497 → 1,000 → 521 → 487 → 417; Fig. 2H), and 403 (1,497 → 1,000 → 521 → 487 → 403; not shown), which contained the whole array of ions arising from cleavages of C-C bonds. Similarly, fragmentations resulting from precursor ion holding charge at C-8 may also lead to the ion series in which the ion of m/z 375 formed by allylic cleavage becomes prominent. The cleavages from the various precursors with various locations of charge site due to proton rearrangement led to the formation of the whole array of ion series with CH2 interval (14 Da).
      By contrast, the MS5 spectrum of m/z 485 arising from a keto PDIM specimen contained the ion series that is 2 Da lighter and is characteristic to the family (discussed subsequently).

      CID MSn on 3-keto, 4-methyl, 9,11-di-(3,5,7-trimethylhexaeicosanoyl) tetratriacontanediol (C29:0/C29:0-kC35:0 PDIM)

      The MS2 spectra of the [M + Na]+, [M + Li]+, and [M+ NH4]+ ions of C29:0/C29:0-kC35:0 PDIM at m/z 1,402.5 (not shown), 1,386.4 (Fig. 3A), 1,397.5 (Fig. 3B), respectively, are similar to those observed for methoxy-PDIM family as shown in Fig. 2. The former spectrum (Fig. 3A) is dominated by the ions of m/z 948 arising from loss of the 29:0-FA substituent at C11, and of m/z 509 arising from further loss of the remaining 29:0-FA at C9 (Scheme 3). The speculation of the preferential loss of FA substituent at C11 is consistent with the observation of the ion of m/z 215 in the MS3 spectrum of the ion of m/z 509 (1,386 → 509; Fig. 3C). This ion is equivalent to the ion of m/z 231 in Fig. 2E, arising from cleavage of allylic bond (Scheme 3) similar to that observed for the methoxy-PDIM family (Scheme 1). The spectrum (Fig. 3C) also contained the ions of m/z 385 (another allylic cleavage), and of m/z 399, 481 (cleavage of C2-CO bond), 491 (509 – H2O), and 423 (β-cleavage with γ-H rearrangement) (Scheme 3), indicating that the ion of m/z 509 may represent a lithiated Δ9, 11 k35:0 phthiocerol. Similar fragmentation processes (Scheme 3) were also seen in the MS3 spectrum of the analogous ion of m/z 481 (Fig. 3E), representing a lithiated Δ9, 11 k-33:0 phthiocerol arising from the [M + Li]+ ion of m/z 1,400 consisting of the 29:0/32:0-k33:0 major isomer together with 29:0/30:0-k35:0 and 27:0/32:0-k35:0 minor isomers (equivalent to the NH4+ adduct ion of m/z 1,411 in Table 1; the structural assignments of the 29:0/32:0-k33:0, 29:0/30:0-k35:0, and 27:0/32:0-k35:0 isomers can be found in supplementary Fig. 2).
      Figure thumbnail gr3
      Fig. 3The MS2 spectra of the [M + Li]+ ion of C29:0/C29:0-kC34:0 PDIM at m/z 1,386.4 (A), MS3 spectra of the ions of m/z 509 (1,386 →509) (C), and of m/z 481 (1,400.5 → 481) (from C29:0/C32:0-kC32:0) (E). Also shown are the MS2 spectra of the corresponding [M + NH4]+ ion at m/z 1,397.5 (B), its MS3 spectrum of the ion of m/z 942 (1,397 → 942) (D), and MS5 spectrum of the ion of m/z 485 (1,397 → 942 → 503 → 485) (F).
      TABLE 1The molecular species of PDIMs ([M + NH4]+) from M. tuberculosis identified by LIT MSn with high-resolution MS
      MeasuredTheoretical MassDeviationRelative IntensityCompositionMajor Structures
      Abundances in the descending order.
      Minor Structures
      Abundances in the descending order.
      DaDamDa%
      1,245.27601,245.27590.110.27C82 H166 O5 N27:0/27:0-m27:0
      Minor isomers not defined.
      1,257.27621,257.27590.320.32C83 H166 O5 N27:0/27:0-k29:0
      Minor isomers not defined.
      1,273.30741,273.30720.230.41C84 H170 O5 N27:0/27:0-m29:0
      Minor isomers not defined.
      1,285.30741,285.307400.67C85 H170 O5 N27:0/27:0-k31:0
      Minor isomers not defined.
      1,299.32351,299.3230.520.71C86 H172 O5 N26:0/29:0-k31:0
      Minor isomers not defined.
      1,313.33911,313.33870.411.52C87 H174 O5 N27:0/27:0-k33:0
      Minor isomers not defined.
      1,327.35431,327.3543−0.045.66C88 H176 O5 N26:0/29:0-k33:027:0/26:0-k35:0
      1,341.37001,341.37000.068.5C89 H178 O5 N27:0/29:0-k33:026:0/30:0-k33:0;26:0/29:0-k34:0; 28:0/26:0-k35:0
      1,355.38561,355.3856−0.0311.82C90 H180 O5 N29:0/29:0-k32:029:0/26:0-k35:0; 27:0/29:0-k34:0; 28:0/29:0-k33:0
      1,357.40301,357.40131.7310.95C90 H182 O5 N27:0/29:0-m33:0
      Minor isomers not defined.
      1,369.40121,369.4013−0.0445.27C91 H182 O5 N29:0/29:0-k33:029:0/27:0-k35:0; 26:0/32:0-k33:0; 26:0/30:0-k35:0: 28:0/28:0-k35:0; 29:0/28:0-k34:0; 30:0/28:0-k33:0; 29:0/30:0-k32:0; 27:0/30:0-k34:0
      1,371.41761,371.41690.6914.47C91 H184 O5 N29:0/26:0-m35:0
      Minor isomers not defined.
      1,383.41701,383.41690.0723.97C92 H184 O5 N29:0/30:0-k33:029:0/29:0-k34:0; 29:0/28:0-k35:0; 29:0/30:0-k33:0; 27:0/32:0-k33:0; 27:0/30:0-k35:0; 26:0/31:0-k35:0; 29:0/31:0-k32:0
      1,385.43271,385.43260.1261.93C92 H186 O5 N29:0/29:0-m33:027:0/29:0-m35:0; 30/26:0-m35:0; 30:0/27:0-m34:0; 30:0/28:0-m33:0; 29:0/30:0-m32:0; 26:0/32:0-m33:0; 27:0/32:0-m32:0
      1,397.43221,397.4326−0.3755.45C93 H186 O5 N29:0/29:0-k35:0;26:0/32:0-k35:0; 27:0/32:0-k34:0; 27:0/31:0-k35:0; 28:0/32:0-k33:0; 29:0/32:0-k32:0; 28:0/30:0-k35:0; 29:0/30:0-k34:0; 30:0/30:0-k33:0; 29:0/31:0-k33
      1,399.44881,399.44820.6327.54C93 H188 O5 N29:0/30:0-m33:0
      Minor isomers not defined.
      1,411.44891,411.44820.6758.04C94 H188 O5 N29:0/32:0-k33:0;29:0/30:0-k35:0; 27:0/32:0-k35:0
      1,413.46411,413.46390.2796.37C94 H190 O5 N29:0/29:0-m35:0
      1,425.46471,425.46390.8421.92C95 H190 O5 N30:0/32:0-k33:0; 29:0/32:0-k34:029:0/31:0-k35:0; 30:0/32:0-k33:0; 30:0/30:0-k35:0; 32:0/28:0-k35:0; 33:0/29:0-k33:0; 27:0/32:0-k36:0; 27:0/33:0-k35:0
      1,427.48061,427.47951.1352.4C95 H192 O5 N29:0/32:0-m33:029:0/30:0-m35:0; 29:0/31:0-m34:0; 29:0/29:0-m35:0
      1,439.47991,439.47950.461.38C96 H192 O5 N29:0/32:0-k35:031:0/32:0-k33:0; 30:0/32:0-k34:0; 31:0/30:0-k35:0
      1,453.49571,453.49520.5334.67C97 H194 O5 N32:0/32:0-k33:0; 30:0/32:0-k35:029:0/32:0-k36:0; 29:0/33:0-k35:0;
      1,455.51141,455.51080.657.3C97 H196 O5 N29:0/32:0-m35:030:0/31:0-m35:0
      1,467.51131,467.51080.499.6C98 H196 O5 N32:0/32:0-k34:0; 31:0/32:0-k35:032:0/31:0-k35:0;
      1,469.52731,469.52650.8629.83C98 H198 O5 N30:0/32:0-m35:032:0/32:0-m33:0
      1,481.52681,481.52650.3235.06C99 H198 O5 N32:0/32:0-k35:0
      Minor isomers not defined.
      1,495.54231,495.54210.213.99C100 H200 O5 N32:0/33:0-k35:0
      Minor isomers not defined.
      1,497.55821,497.55780.434.11C100 H202 O5 N32:0/32:0-m35:0
      Minor isomers not defined.
      1,509.55831,509.55780.581.41C101 H202 O5 N32:0/34:0-k35:0
      Minor isomers not defined.
      1,511.57551,511.57342.072.74C101 H204 O5 N32:0/33:0-m35:0
      Minor isomers not defined.
      1,523.57421,523.57340.781.84C102 H204 O5 N33:0/34:0-k35:0
      Minor isomers not defined.
      1,539.60511,539.60470.441.62C103 H208 O5 N32:0/35:0-m35:0
      Minor isomers not defined.
      a Abundances in the descending order.
      b Minor isomers not defined.
      The MS2 spectrum of the [M+ NH4]+ ions at m/z 1,397 (Fig. 3B), again, contained the prominent ion of m/z 941.9, arising from expulsion of 29:0-FA and NH3, and further loss of the remaining 29:0-FA gave rise to the ion of m/z 503 (Fig. 3D), in which the charge site is relocated at the carbonyl group Scheme 4). The ion at m/z 485 arose from additional loss of a water molecule (Fig. 3D), probably involving a prior 1,6-H shift to yield a carbonium ion with 3-OH side chain, whose charge may reside at C-7. This is followed by loss of a water molecule to form a stable allylic carbonium ion of m/z 485 with the participation of the adjacent hydrogen at C-4 (Scheme 4). These fragmentation processes are supported by the MS3 spectrum of the ion of m/z 941.9 (1,397 → 942; Fig. 3D), and the MS4 spectrum of the ion of m/z 503 (not shown).
      Further dissociation of the ion of m/z 485 (1,397 → 941 → 503 → 485; Fig. 3F) gave rise to the ion series of m/z 457, 443, 429, 415, and 401, … , and so forth, in which the ion of m/z 457 is likely arising from loss of an ethane residue, in contrast to loss of a C3-alkane observed for the ion of m/z 487 originated from a methoxy PDIM compound as shown in Fig. 2G. The spectrum also contained the ion series of m/z 403, 389, 375, 361, and so forth, consistent with the notion that elimination of a water molecule involves the participation of the hydrogen at C-4.

      Characterization of M. tuberculosis biofilm PDIMs as [M + NH4]+ ions

      A previous report indicates that M. tuberculosis produced mainly diesters of phthiocerol A and phthiodiolone A, and phthiocerol B diesters were present in very small amounts so that their detection was unreliable (
      • Minnikin D.E.
      • Dobson G.
      • Goodfellow M.
      • Magnusson M.
      • Ridella M.
      Distribution of some mycobacterial waxes based on the phthiocerol family.
      ). High-resolution mass measurements (supplementary Fig. 3) on the NH4+ adduct ion of these mycobacterial waxes clearly resolved phthiodiolone A dimycolates, which gave elemental compositions of C86H168(CH2)nO5NH4 (n = 1, 2, … , 12), from phthiocerol A dimycolates having elemental compositions of C86H170(CH2)nO5 NH4 (Table 1). More than one isomer was found in most of the species, and molecules in the phthiocerol B family were not detected. Examples for complete characterization of the entire PDIM family from biofilm of M. tuberculosis (Table 1) applying LIT MSn are given below.
      Fig. 4A showed the MS2 spectrum of the [M+ NH4]+ ions of m/z 1,385.4, which is dominated by m/z 929.9 arising from loss of 29:0-FA acid substituent as NH4+ salt (455.5 Da). Further dissociation of the ion of m/z 929 (1,385 → 929; Fig. 4B) gave rise to ions at m/z 491 arising from loss of the remaining 29:0-FA substituent (438.5 Da), along with m/z 459 (491 – CH3OH) and 897.9 (929.9 – CH3OH) arising from further loss of CH3OH. The results indicate that the molecule belongs to the PDIM family consisting of a major 29:0/29:0-m33:0 species. The spectrum also contains the 519/487 ion pair, signifying the presence of 27:0-FA/m33:0 phthiocerol substituents and the presence of 29:0/27:0-m35:0-PDIM isomer. The recognition of this latter isomer is further supported by observation of the ion of m/z 958.1 arising from loss of 27:0-FA in Fig. 4A. The MS3 spectrum of m/z 958 (1,385 → 958; Fig. 4C) contained the m/z 519/487 ion pairs indicating the presence of 29:0-FA/m35:0 phthiocerol substituents, consistent with the assignment of 29:0/27:0-m35:0-PDIM.
      Figure thumbnail gr4
      Fig. 4The MS2 spectrum of the [M + NH4]+ ion at m/z 1,385.4 (A), it MS3 spectra of the ions of m/z 930 (1,385 → 930) (B), of m/z 958 (1,385 → 958) (C), of m/z 916 (1,385 → 916) (D), of m/z 972 (1,385 → 972) (E), and its MS4 spectrum of the ions of m/z 459 (1,385 →930 →459) (F).
      In Fig. 4A, ions at m/z 915.9, arising from loss of 30:0-FA acid substituent, were also observed. MS3 on the ions of m/z 915.9 (1,385 → 915; Fig. 4D) yielded ion pairs of 519/487, 505/473, 491/459, 477/445, reflecting the presence of 26:0/m35:0, 27:0/m34:0, 28:0/m33:0, 29:0/m32:0 substituents, respectively. The results indicate that the ions of m/z 1,385 also represent 30:0/26:0-m35:0, 30:0/27:0-m34:0, 30:0/28:0-m33:0, and 30:0/29:0-m32:0 minor isomers. The assignment of 30:0/26:0-m35:0 isomer is consistent with the observation of the ion of m/z 972.1, arising from loss of 26:0-FA substituent in Fig. 4A. The MS3 spectrum of m/z 972 (1,385 → 972; Fig. 4E) contained the ion pairs of m/z 519/487 and of 491/459, revealing the 30:0/m35:0 and 32:0/m33:0 substituents of the molecules, respectively, and the assignment of 26:0/30:0-m35:0 and 26:0/32:0-m33:0 isomers. The assignment of 26:0/30:0-m35:0 isomer is consistent with the earlier identification of 30:0/26:0-m35:0 isomer (in this assignment, fatty acyl groups at C9 and C11 are not specified). The identification of the 26:0/32:0-m33:0 isomer is also consistent with the observation of the ion of m/z 897 in Fig. 4A, arising from loss of 32:0-FA substituent, and is further supported by the MS3 spectrum of the ion of m/z 897 (1,385 → 897; data not shown), which contains the m/z 491/459 and 477/445 ion pairs.
      The profiles of the MS4 spectra of the ions of m/z 487 (1,385 →958 →487; not shown), 473 (1,385 →958 →473; not shown), 459 (1,385 →930 →459; Fig. 4F), and 445 (1,385 →915 →445; not shown) are similar to that shown in Fig. 2G, supporting the notion that they represent the demethoxylated C35-, C34-. C33-, and C32-phthiocerol ions with conjugated double bond (Scheme 1).
      Similar approaches were also applied to reveal the structural complexity of phthiodiolone dimycerosate family of the extract (Table 1). For example, the MS2 spectrum of the [M+ NH4]+ ion at m/z 1,411.5 (Fig. 5A) contained major ions at m/z 956 and 914 arising from losses of NH3 and 29:0- and 32:0-mycosanoic acids respectively, indicating the linkage of 29:0- and 32:0-mycosanoic acids to 9- and 11- of the phthiodiolone backbone. Further dissociation of the ion of m/z 956 (1,411 → 956; Fig. 5B) gave rise to ions at m/z 475, arising from loss of the remaining 32:0-acid, consistent with the presence of the protonated 32:0-mycosanoic acid ion at m/z 481. The spectrum also consisted of the ion of m/z 457 arising from further loss of water from m/z 475. This loss of water is supported by the MS4 spectrum of the ion of m/z 475 (1,411 → 956 → 475; data not shown), which is dominated by the ion of m/z 457, indicating that the molecule belongs to the phthiodiolone family. The above structural information led to assignment of the major 29:0/32:0-k33:0 structure. Because the spectrum (Fig. 5B) also contained the ions of m/z 503 (loss of 30:0-FA) and 485 (503 – H2O), a 29:0/30:0-k35:0 isomeric structure can be defined. MS3 on the ion of m/z 914 (1,411 → 914; Fig. 5C) gave rise to the major ions of m/z 475 and 457 (475 – H2O), arising from loss of 29:0-FA substituent, consistent with the structural assignment of the 29:0/32:0-k33:0 isomer. The spectrum also contained the minor ions of m/z 503 and 485 (503 – H2O), arising from loss of 27:0-FA substituent, pointing to the presence of 27:0/32:0-k35:0 minor isomer.
      Figure thumbnail gr5
      Fig. 5The MS2 spectrum of the [M + NH4]+ ion at m/z 1,411.5 (A), its MS3 spectra of the ion of m/z 956 (1,411 → 956) (B), of m/z 914 (1,411 → 914) (C), of m/z 984 (1,411 → 984) (D), of m/z 942 (1,411 → 942) (E), and of the MS5 spectrum of the ion of m/z 457 (1,411 → 914 → 475 → 457) (F).
      In Fig. 5A, ions at m/z 984 and 942, arising from loss of 27:0- and 30:0-FA substituents, respectively, were also observed. Further dissociation of the ion of m/z 984 (1,411 → 984; Fig. 5D) gave rise to ions of m/z 503 and 485 (503 – H2O), defining the 32:0-FA substituent. These results further support the earlier assignment of the 27:0/32:0-k35:0 isomer. MS3 on the ion of m/z 942 (1,411 → 942; Fig. 5E) also yielded ions of m/z 503 and 485 (503 – H2O), indicating the presence of 29:0-FA substituent, and the k35:0-phthiodiolone chain, and confirming the assigned 29:0/30:0-k35:0 structure as described earlier.
      The MS5 spectra of the ions of m/z 485 stemming from m/z 942 (1,411 → 942 → 503 → 485), 984 (1,411 → 984 → 503 → 485), and 956 (1,411 → 956 → 503 → 485) are identical to that shown in Fig. 3D, and the profiles of the MS5 spectra of the ions of m/z 457 originating from m/z 914 (1,411 → 914 → 475 → 457; Fig. 5F) and 956 (1,411 → 956 → 475 → 457; not shown) are also identical. These spectra comprise the ion series defining the k35- and k33:0- phthiodiolone chains, respectively. These results led to the notion that the ion of m/z 1,411 consists of a major 29:0/32:0-k33:0 isomer together with minor isomers of 27:0/32:0-k35:0 and 29:0/30:0-k35:0 (Table 1).

      Characterization of multiple-methyl-branched long-chain mycocerosic (mycoceranic) acid substituents

      To assign the structure of mycoceranic acid substituents, released free acids by alkaline hdrolysis were converted to the AMPP derivatives and subjected to ESI MSn analysis in the positive-ion mode. Three major species were observed at m/z 647, 619, and 605, corresponding to C32-, C30-, and C29-FA AMPP derivatives, respectively. The MS2 spectra of m/z 647 (Fig. 6A), 619 (Fig. 6B), and 605 (Fig. 6C) all contained abundant ions at m/z 183 and 169, which are characteristic of FA-AMPP derivatives (
      • Bollinger J.G.
      • Rohan G.
      • Sadilek M.
      • Gelb M.H.
      LC/ESI-MS/MS detection of FAs by charge reversal derivatization with more than four orders of magnitude improvement in sensitivity.
      ,
      • Bollinger J.G.
      • Thompson W.
      • Lai Y.
      • Oslund R.C.
      • Hallstrand T.S.
      • Sadilek M.
      • Turecek F.
      • Gelb M.H.
      Improved sensitivity mass spectrometric detection of eicosanoids by charge reversal derivatization.
      ,
      • Yang K.
      • Dilthey B.G.
      • Gross R.W.
      Identification and quantitation of fatty acid double bond positional isomers: a shotgun lipidomics approach using charge-switch derivatization.
      ,
      • Wang M.
      • Han R.H.
      • Han X.
      Fatty acidomics: global analysis of lipid species containing a carboxyl group with a charge-remote fragmentation-assisted approach.
      ,
      • Gross M.L.
      Charge-remote fragmentations: method, mechanism and applications.
      ). The MS2 spectrum of the ion of m/z 647 (Fig. 6A) also contained the ion series at m/z 365, 323, 281, and 239, along with the ion series of m/z 351, 295, and 253, pointing to the position of the methyl side chains at C-2, 4, 6, and 8 of 32:0-mycoceranic acid (see insets for fragmentation pathways), and gave assignment of 2,4,6,8-tetramethyl-octaeicosanoic acid structure. The MS2 spectrum of the ion of m/z 619 (Fig. 6B) contained the similar ion series, indicating the presence of 2, 4, 6, 8-tetramethyl-hexaeicosanoic acid (30:0-mycoceranic acid). In contrast, the MS2 spectrum of the ion of m/z 605 (Fig. 6C) contained the ion series of m/z 323, 281, and 239, along with the ion pair of m/z 295 and 253, indicating the presence of a 2,4,6-trimethyl-hexaeicosanoic acid (29:0-mycoceranic acid) structure.
      Figure thumbnail gr6
      Fig. 6The MS2 spectra of the PDIM alkaline hydrolysate-AMPP derivative of the ions of m/z 649 (A), of m/z 619 (B), and of m/z 605 (C). The fragmentation pathways leading to locating the position of the methyl side chains of the FA substituents (subset) are also shown.

      DISCUSSION

      PDIM formed alkaline metal adduct ions ([M+ Alk]+; Alk = Li, Na, K) and [M + NH4 ]+ ions when subjected to ESI in the presence of Li+, Na+, K+, or NH4+. ESI LIT MSn with high-resolution MS on these adduct ions affords a near complete structural determination of PDIMs, revealing the presence of many homologous and isomeric structures (Table 1). Elemental compositions derived from high-resolution MS readily distinguish methoxy-PDIM and keto-PDIM molecules, while MSn on the [M+ Alk]+ adduct ions (Alk = Li, Na) and [M + NH4 ]+ provide spectroscopic evidence for unambiguous distinction of this two families via the specific neutral losses (i.e., methanol vs. water loss). The more facile cleavages of the mycoceranic acid substituent at C11 than that at C9 upon CID as observed in this study may be applicable for specifically defining the mycoceranic acid substituents on the phthiocerol backbone (at C9 or C11). However, more studies with standard compounds with two different mycoceranic acid chains are required to confirm this finding. In the negative-ion mode, in contrast, the MSn spectra obtained from the [M + X] adduct ions do not provide sufficient informative ions applicable for structure identification, despite that ions in the fashions of [M + X] (X = Cl, HCO2) are readily formed, and the elemental compositions derived from high-resolution MS are also distinguishable among the methoxy-PDIM and keto-PDIM families (data not shown).
      Fragment ions arising from cleavages of C-C bonds along the phthiocerol backbone following loss of the mycoceranic acid substituents may involve the precursors in various resonance forms differed by the charge sites due to proton delocalization (
      • Wysocki V.H.
      • Tsaprailis G.
      • Smith L.L.
      • Breci L.A.
      Mobile and localized protons: a framework for understanding peptide dissociation.
      ,
      • Paizs B.
      • Suhai S.
      Fragmentation pathways of protonated peptides.
      ). Thus, charge-remote fragmentation processes may not be invoked (
      • Gross M.L.
      Charge-remote fragmentations: method, mechanism and applications.
      ,
      • Gross M.L.
      Charge-remote fragmentation: an account of research on mechanisms and applications.
      ), and other fragmentation processes become available (
      • Harvey D.J.
      A new charge-associated mechanism to account for the production of fragment ions in the high-energy CID spectra of fatty acids.
      ). The stark differences between the MS5 spectra of m/z 485 (Fig. 3F) and of m/z 487 (Fig. 2G) lies on the notion that the former spectrum contains abundant ions at m/z 191, 177, 163, 149, 135, and so forth; while the analogous ions seen at m/z 193, 179, 165, 151, 137, and so forth in the latter spectrum are less prominent (Fig. 3F). The prominence of the ion series of m/z 191, 177, 163, 149, and so forth, arising from m/z 485 (Fig. 3F) may be attributable to the fact these ions consist of one more double bond than the analogous ions of m/z 193, 179, 165, and so forth, arising from m/z 487 and are more conjugated and more stable. Striking differences were also observed for the MS5 spectra of the ions of m/z 459 arising from m34:0 (Fig. 4F) and of 457 arising from k33:0 backbones (Fig. 5F). These differences in the profiles of the MSn spectra between those arising from phthiocerol and from phthiodiolone families also provide useful information for their structural differentiation by MS.
      The advantage of characterization of mycoceranic acid substituents using charge-switch formation of the FA-AMPP derivatives is that feature ions of m/z 183 and 169 are readily recognizable (Fig. 6) (
      • Tatituri R.V.
      • Wolf B.
      • Brenner M.
      • Turk J.
      • Hsu F-F.
      Characterization of polar lipids of Listeria monocytogenes by HCD and low-energy CAD linear ion-trap mass spectrometry with electrospray ionization.
      ,
      • Bollinger J.G.
      • Rohan G.
      • Sadilek M.
      • Gelb M.H.
      LC/ESI-MS/MS detection of FAs by charge reversal derivatization with more than four orders of magnitude improvement in sensitivity.
      ,
      • Bollinger J.G.
      • Thompson W.
      • Lai Y.
      • Oslund R.C.
      • Hallstrand T.S.
      • Sadilek M.
      • Turecek F.
      • Gelb M.H.
      Improved sensitivity mass spectrometric detection of eicosanoids by charge reversal derivatization.
      ,
      • Yang K.
      • Dilthey B.G.
      • Gross R.W.
      Identification and quantitation of fatty acid double bond positional isomers: a shotgun lipidomics approach using charge-switch derivatization.
      ,
      • Wang M.
      • Han R.H.
      • Han X.
      Fatty acidomics: global analysis of lipid species containing a carboxyl group with a charge-remote fragmentation-assisted approach.
      ), and ions from charge-remote fragmentations can locate the methyl side chains (
      • Tatituri R.V.
      • Wolf B.
      • Brenner M.
      • Turk J.
      • Hsu F-F.
      Characterization of polar lipids of Listeria monocytogenes by HCD and low-energy CAD linear ion-trap mass spectrometry with electrospray ionization.
      ,
      • Wang M.
      • Han R.H.
      • Han X.
      Fatty acidomics: global analysis of lipid species containing a carboxyl group with a charge-remote fragmentation-assisted approach.
      ) and the functional groups unambiguously (Fig. 6, inset). By contrast, the traditional GC/MS method for identification of mycocerosic acids is laborious, requiring acid methanolysis to form the methyl ester, separated by TLC, followed by reductive degradation to mycocerosic alcohol and another two-dimensional TLC purification, before formation of the final t-butyldimethylsilyl ether derivatives (
      • Alibaud L.
      • Rombouts Y.
      • Trivelli X.
      • Burguière A.
      • Cirillo S.L.G.
      • Cirillo J.D.
      • Dubremetz J-F.
      • Guérardel Y.
      • Lutfalla G.
      • Kremer L.
      A Mycobacterium marinum TesA mutant defective for major cell wall-associated lipids is highly attenuated in Dictyostelium discoideum and zebrafish embryos.
      ,
      • Minnikin D.E.
      • Dobson G.
      • Hutchinson I.G.
      Charac­terization of phthiocerol dimycocerosates from Mycobacterium tuberculosis.
      ,
      • Dobson G.
      • Minnikin D.E.
      • Besra G.S.
      • Mallet A.I.
      • Magnusson M.
      Characterisation of phenolic glycolipids from Mycobacterium marinum.
      ). The superb sensitivity gained for the acid detected as the AMPP derivative (as compared with its underivatized form) also facilitates structural identification and quantitation (
      • Tatituri R.V.
      • Wolf B.
      • Brenner M.
      • Turk J.
      • Hsu F-F.
      Characterization of polar lipids of Listeria monocytogenes by HCD and low-energy CAD linear ion-trap mass spectrometry with electrospray ionization.
      ,
      • Bollinger J.G.
      • Rohan G.
      • Sadilek M.
      • Gelb M.H.
      LC/ESI-MS/MS detection of FAs by charge reversal derivatization with more than four orders of magnitude improvement in sensitivity.
      ,
      • Bollinger J.G.
      • Thompson W.
      • Lai Y.
      • Oslund R.C.
      • Hallstrand T.S.
      • Sadilek M.
      • Turecek F.
      • Gelb M.H.
      Improved sensitivity mass spectrometric detection of eicosanoids by charge reversal derivatization.
      ,
      • Yang K.
      • Dilthey B.G.
      • Gross R.W.
      Identification and quantitation of fatty acid double bond positional isomers: a shotgun lipidomics approach using charge-switch derivatization.
      ,
      • Wang M.
      • Han R.H.
      • Han X.
      Fatty acidomics: global analysis of lipid species containing a carboxyl group with a charge-remote fragmentation-assisted approach.
      ). This aspect may deserve further investigation.
      The insight into the detailed structures of the PDIMs (Table 1) in this study also permits the realization of the structure similarities among the methoxy- and keto-PDIM families. For example, the major structures of the ions of m/z 1,369 (29:0/29:0-k30:0) and 1,385 (29:0/29:0-m30:0) all contained 29:0/29:0 substituents; the FA substituents in the species of m/z 1,411 (29:0/32:0-k30:0) of keto family and of m/z 1,427 (29:0/32:0-m30:0) in the methoxy family are all 29:0/32:0. The ESI/MS profiles of these two families are also similar (see supplementary Fig. 2C, D). The results are consistent with the notion that methoxy-PDIM is formed from keto-PDIM (
      • Cox J.S.
      • Chen B.
      • McNeil M.
      • Jacobs W.R.
      Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice.
      ,
      • Siméone R.
      • Constant P.
      • Guilhot C.
      • Daffé M.
      • Chalut C.
      Identification of the missing trans-acting enoyl reductase required for phthiocerol dimycocerosate and phenolglycolipid biosynthesis in Mycobacterium tuberculosis.
      ,
      • Gastambide Odier M.
      • Delaumeny J.M.
      • Lederer E.
      Biosynthesis of phthiocerol; incorporation of methionine and propionic acid.
      ). It is also notable that keto-PDIMs are more prominent than the methoxy-PDIM species, while phthiodiolone dimycocerosates were the minor components previously reported in the M. tuberculosis cells (
      • Siméone R.
      • Constant P.
      • Guilhot C.
      • Daffé M.
      • Chalut C.
      Identification of the missing trans-acting enoyl reductase required for phthiocerol dimycocerosate and phenolglycolipid biosynthesis in Mycobacterium tuberculosis.
      ).

      REFERENCES

        • Asselineau J.
        Sur quelques substances ii 60 atomes de carbone isolees des lipides de souches humaines de Mycobacterium tuberculosis.
        Bull. Soc. Chim. Fr. 1954; 21: 108-112
        • Noll H.
        The chemistry of some native constituents of the purified wax of Mycobacterium tuberculosis.
        J. Biol. Chem. 1957; 224: 149-164
        • Reeves R.E.
        • Anderson R.J.
        The chemistry of the lipids of tubercle bacilli: XLVIII. The occurrence of phthiocerol in the wax from various strains of the human tubercle bacillus.
        J. Biol. Chem. 1937; 119: 535-541
        • Stodola F.H.
        • Anderson R.
        The chemistry of the lipids of tubercle bacilli: XLVI. Phthiocerol, a new alcohol from the wax of the human tubercle bacillus.
        J. Biol. Chem. 1936; 114: 467-472
        • Redman J.E.
        • Shaw M.J.
        • Mallet A.I.
        • Santos A.L.
        • Roberts C.A.
        • Gernaey A.M.
        • Minnikin D.E.
        Mycocerosic acid biomarkers for the diagnosis of tuberculosis in the Coimbra Skeletal Collection.
        Tuberculosis (Edinb.). 2009; 89: 267-277
        • Minnikin D.E.
        • Kremer L.
        • Dover L.G.
        • Besra G.S.
        The methyl-branched fortifications of Mycobacterium tuberculosis.
        Chem. Biol. 2002; 9: 545-553
        • Onwueme K.C.
        • Vos C.J.
        • Zurita J.
        • Ferreras J.A.
        • Quadri L.E.N.
        The dimycocerosate ester polyketide virulence factors of mycobacteria.
        Prog. Lipid Res. 2005; 44: 259-302
        • Yu J.
        • Tran V.
        • Li M.
        • Huang X.
        • Niu C.
        • Wang D.
        • Zhu J.
        • Wang J.
        • Gao Q.
        • Liu J.
        Both phthiocerol dimycocerosates and phenolic glycolipids are required for virulence of Mycobacterium marinum.
        Infect. Immun. 2012; 80: 1381-1389
        • Daffé M.
        • Laneelle M.A.
        Distribution of phthiocerol diester, phenolic mycosides and related compounds in mycobacteria.
        J. Gen. Microbiol. 1988; 134: 2049-2055
        • Minnikin D.E.
        • Dobson G.
        • Goodfellow M.
        • Magnusson M.
        • Ridella M.
        Distribution of some mycobacterial waxes based on the phthiocerol family.
        J. Gen. Microbiol. 1985; 131: 1375-1381
        • Reed M.B.
        • Domenech P.
        • Manca C.
        • Su H.
        • Barczak A.K.
        • Kreiswirth B.N.
        • Kaplan G.
        • Barry C.E.
        A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response.
        Nature. 2004; 431: 84-87
        • Kolattukudy P.E.
        • Fernandes N.D.
        • Azad A.K.
        • Fitzmaurice A.M.
        • Sirakova T.D.
        Biochemistry and molecular genetics of cell-wall lipid biosynthesis in mycobacteria.
        Mol. Microbiol. 1997; 24: 263-270
        • Rousseau C.
        • Winter N.
        • Pivert E.
        • Bordat Y.
        • Neyrolles O.
        • Avé P.
        • Huerre M.
        • Gicquel B.
        • Jackson M.
        Production of phthiocerol dimycocerosates protects Mycobacterium tuberculosis from the cidal activity of reactive nitrogen intermediates produced by macrophages and modulates the early immune response to infection.
        Cell. Microbiol. 2004; 6: 277-287
        • Cox J.S.
        • Chen B.
        • McNeil M.
        • Jacobs W.R.
        Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice.
        Nature. 1999; 402: 79-83
        • Camacho L.R.
        • Ensergueix D.
        • Perez E.
        • Gicquel B.
        • Guilhot C.
        Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis.
        Mol. Microbiol. 1999; 34: 257-267
        • Camacho L.R.
        • Constant P.
        • Raynaud C.
        • Lanéelle M-A.
        • Triccas J.A.
        • Gicquel B.
        • Daffé M.
        • Guilhot C.
        Analysis of the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis: evidence that this lipid is involved in the cell wall permeability barrier.
        J. Biol. Chem. 2001; 276: 19845-19854
        • Murry J.P.
        • Pandey A.K.
        • Sassetti C.M.
        • Rubin E.J.
        Phthiocerol dimycocerosate transport is required for resisting interferon-γ–independent immunity.
        J. Infect. Dis. 2009; 200: 774-782
        • Cambier C.J.
        • Takaki K.K.
        • Larson R.P.
        • Hernandez R.E.
        • Tobin D.M.
        • Urdahl K.B.
        • Cosma C.L.
        • Ramakrishnan L.
        Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids.
        Nature. 2014; 505: 218-222
        • Goren M.B.
        • Brennan P.J.
        Mycobacterial lipids: chemistry and biologic activities. In Tuberculosis.
        Saunders Company. 1979; : 63-193
        • Minnikin D.E.
        • Goodfellow M.
        Lipid Com­position in the Classification and Identification of Acid-Fast Bacteria.
        Academic. 1980;
        • Smith D.W.
        • Randall H.M.
        • Gastambide-Odier M.M.
        • Koevoet A.L.
        The characterization of mycobacterial strains by the composition of their lipide extracts.
        Ann. N. Y. Acad. Sci. 1957; 69: 145-157
        • Demartreau-Ginsburg H.
        • Lederer E.
        • Ryhage R.
        • Stallberg-Stenhagen S.
        • Stenhagen E.
        Structure of phthiocerol.
        Nature. 1959; 183: 117-119
        • Alibaud L.
        • Rombouts Y.
        • Trivelli X.
        • Burguière A.
        • Cirillo S.L.G.
        • Cirillo J.D.
        • Dubremetz J-F.
        • Guérardel Y.
        • Lutfalla G.
        • Kremer L.
        A Mycobacterium marinum TesA mutant defective for major cell wall-associated lipids is highly attenuated in Dictyostelium discoideum and zebrafish embryos.
        Mol. Microbiol. 2011; 80: 919-934
        • Minnikin D.E.
        • Dobson G.
        • Hutchinson I.G.
        Charac­terization of phthiocerol dimycocerosates from Mycobacterium tuberculosis.
        Biochim. Biophys. Acta. 1983; 753: 445-449
        • Dobson G.
        • Minnikin D.E.
        • Besra G.S.
        • Mallet A.I.
        • Magnusson M.
        Characterisation of phenolic glycolipids from Mycobacterium marinum.
        Biochim. Biophys. Acta. 1990; 1042: 176-181
        • Nicoara S.C.
        • Minnikin D.E.
        • Lee O.C.Y.
        • O'Sullivan D.M.
        • McNerney R.
        • Pillinger C.T.
        • Wright I.P.
        • Morgan G.H.
        Development and optimization of a gas chromatography/mass spectrometry method for the analysis of thermochemolytic degradation products of phthiocerol dimycocerosate waxes found in Mycobacterium tuberculosis.
        Rapid Commun. Mass Spectrom. 2013; 27: 2374-2382
        • Daffé M.
        • Draper P.
        The envelope layers of mycobacteria with reference to their pathogenicity In Advances in Microbial Physiology.
        Academic Press. 1997; : 131-203
        • Daffé M.
        • Lacave C.
        • Lanéelle M.-A.
        • Lanéelle G.
        Structure of the major triglycosyl phenol-phthiocerol of Mycobacterium tuberculosis (strain Canetti).
        Eur. J. Biochem. 1987; 167: 155-160
        • Constant P.
        • Perez E.
        • Malaga W.
        • Lanéelle M-A.
        • Saurel O.
        • Daffé M.
        • Guilhot C.
        Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex: evidence that all strains synthesize glycosylated p-hydroxybenzoic methyl esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene.
        J. Biol. Chem. 2002; 277: 38148-38158
        • Siméone R.
        • Constant P.
        • Guilhot C.
        • Daffé M.
        • Chalut C.
        Identification of the missing trans-acting enoyl reductase required for phthiocerol dimycocerosate and phenolglycolipid biosynthesis in Mycobacterium tuberculosis.
        J. Bacteriol. 2007; 189: 4597-4602
        • Jain M.
        • Petzold C.J.
        • Schelle M.W.
        • Leavell M.D.
        • Mougous J.D.
        • Bertozzi C.R.
        • Leary J.A.
        • Cox J.S.
        Lipidomics reveals control of Mycobacterium tuberculosis virulence lipids via metabolic coupling.
        Proc. Natl. Acad. Sci. USA. 2007; 104: 5133-5138
        • Casas-Arce E.
        • ter Horst B.
        • Feringa B.L.
        • Minnaard A.J.
        Asymmetric total synthesis of PDIM A: a virulence factor of Mycobacterium tuberculosis.
        Chemistry. 2008; 14: 4157-4159
        • Ojha A.K.
        • Baughn A.D.
        • Sambandan D.
        • Hsu T.
        • Trivelli X.
        • Guerardel Y.
        • Alahari A.
        • Kremer L.
        • Jacobs W.R.
        • Hatfull G.F.
        Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria.
        Mol. Microbiol. 2008; 69: 164-174
        • Tatituri R.V.
        • Wolf B.
        • Brenner M.
        • Turk J.
        • Hsu F-F.
        Characterization of polar lipids of Listeria monocytogenes by HCD and low-energy CAD linear ion-trap mass spectrometry with electrospray ionization.
        Anal. Bioanal. Chem. 2015; 407: 2519-2528
        • Stendal N.
        The presence of a glycol in the wax of tubercle bacilli.
        C. R. Hebd. Seances Acad. Sci. Ser. D. 1934; 198: 1549-1550
        • Hsu F-F.
        • Turk J.
        Electrospray ionization multiple-stage linear ion-trap mass spectrometry for structural elucidation of triacylglycerols: assignment of fatty acyl groups on the glycerol backbone and location of double bonds.
        J. Am. Soc. Mass Spectrom. 2010; 21: 657-669
        • Wysocki V.H.
        • Tsaprailis G.
        • Smith L.L.
        • Breci L.A.
        Mobile and localized protons: a framework for understanding peptide dissociation.
        J. Mass Spectrom. 2000; 35: 1399-1406
        • Paizs B.
        • Suhai S.
        Fragmentation pathways of protonated peptides.
        Mass Spectrom. Rev. 2005; 24: 508-548
        • Hsu F.F.
        • Turk J.
        Elucidation of the double-bond position of long-chain unsaturated fatty acids by multiple-stage linear ion-trap mass spectrometry with electrospray ionization.
        J. Am. Soc. Mass Spectrom. 2008; 19: 1673-1680
        • Bollinger J.G.
        • Rohan G.
        • Sadilek M.
        • Gelb M.H.
        LC/ESI-MS/MS detection of FAs by charge reversal derivatization with more than four orders of magnitude improvement in sensitivity.
        J. Lipid Res. 2013; 54: 3523-3530
        • Bollinger J.G.
        • Thompson W.
        • Lai Y.
        • Oslund R.C.
        • Hallstrand T.S.
        • Sadilek M.
        • Turecek F.
        • Gelb M.H.
        Improved sensitivity mass spectrometric detection of eicosanoids by charge reversal derivatization.
        Anal. Chem. 2010; 82: 6790-6796
        • Yang K.
        • Dilthey B.G.
        • Gross R.W.
        Identification and quantitation of fatty acid double bond positional isomers: a shotgun lipidomics approach using charge-switch derivatization.
        Anal. Chem. 2013; 85: 9742-9750
        • Wang M.
        • Han R.H.
        • Han X.
        Fatty acidomics: global analysis of lipid species containing a carboxyl group with a charge-remote fragmentation-assisted approach.
        Anal. Chem. 2013; 85: 9312-9320
        • Gross M.L.
        Charge-remote fragmentations: method, mechanism and applications.
        Int. J. Mass Spectrom. Ion Process. 1992; 118–119: 137-165
        • Gross M.L.
        Charge-remote fragmentation: an account of research on mechanisms and applications.
        Int. J. Mass Spectrom. 2000; 200: 611-624
        • Harvey D.J.
        A new charge-associated mechanism to account for the production of fragment ions in the high-energy CID spectra of fatty acids.
        J. Am. Soc. Mass Spectrom. 2005; 16: 280-290
        • Gastambide Odier M.
        • Delaumeny J.M.
        • Lederer E.
        Biosynthesis of phthiocerol; incorporation of methionine and propionic acid.
        Chem. Ind. 1963; 31: 1285-1286