Introduction

Listeria monocytogenes (L. monocytogenes), a Gram-positive, food-borne bacterium, is one of the most virulent food-borne pathogens that cause listeriosis. L. monocytogenes can be isolated from many foods, including raw and ready-to-eat meat, poultry, egg, seafood, and vegetables and can grow vigorously at refrigerator temperatures and in osmotically stressful environments with high salt concentrations. Listeria is ubiquitous in the environment and is primarily transmitted via the oral route after ingestion of contaminated food products, after which the organism can penetrate the intestinal tract, grow, and reproduce inside the host’s cells to cause systemic infections [1–5]. The subject of listeriosis caused by L. monocytogenes is beyond the scope of this study. Interested readers refer to the following reviews for detail [6, 7].

Lipid plays major role in Listeria survival, which depends on membrane lipid homeostasis and its ability to adjust lipid composition to accommodate to different environments. L. monocytogenes cell exposure to acid stress at low pH, such as in presence of hydrochloric, acetic, lactic, and benzoic acids [8], and to the sanitizer benzalkonium chloride [9] alters the composition of polar and neutral lipids. Acid adaptation in L. monocytogenes was correlated with a decrease in total phospholipids including cardiolipin, phosphatidylglycerol, phosphoaminolipid, and phosphatidylinositol, reflecting a higher content of the neutral lipid class [8]. Coordinated regulation of cold-induced changes in fatty acids with cardiolipin and phosphatidylglycerol composition among phospholipid species was also reported [10].

Despite a vast body of literatures on the studies of the pathogens of L. monocytogenes related to the changes of the fatty acid profiles in the membrane after stress has been published [10–14], very few have been focused on the structural characterization of the lipids in Listeria. Fischer et al. used FAB/MS and GC/MS combined with chemical reactions to identify the polar lipid species containing lysylcardiolipin in Listeria [15–17]. However, the methods are not sensitive and inherit the shortcomings of FAB/MS, such as the high background ions from FAB matrix that complicate the analysis. Herein, we describe linear ion-trap multiple-stage mass spectrometric approaches with high-resolution mass spectrometry toward structural characterization of the polar membrane lipids, including phosphatidylglycerol, cardiolipins, lysyl cardiolipins, and diglucosyldiacylglycerol that were desorbed as the [M–H]− ions by electrospray ionization. We also adopted a charge-reversed strategy that detects the acids in the positive ion mode by conversion of the free fatty acid to the N-(4-aminomethylphenyl) pyridinium (AMPP) derivative [18–21], following alkaline hydrolysis of the lipid extract to release the fatty acid substituents. These mass spectrometric approaches afford a nearly complete structural characterization of these lipid families, including the location of the methyl branches of the fatty acyl chains.

Materials and methods

Growth and lipid extraction of L. monocytogenes

L. monocytogenes strain 10403S (a gift from H. Shen, University of Pennsylvania) was inoculated into brain-heart infusion (BHI) broth (BD Biosciences) supplemented with 200 μg/ml streptomycin (Sigma-Aldrich) (BHI-STR) and grown overnight at 37 °C. The next day, 4-L flasks each containing 2.5 L of prewarmed BHI-STR broth were inoculated at approximately 1:420 v/v and grown until mid-log phase. Mid-log bacteria were collected by centrifugation, washed in PBS, and then lyophilized for 48 h. After lyophilization, pellets of Listeria were transferred to glass or teflon-coated bottles and crude polar and nonpolar lipids were extracted as previously described [22]. Lipids were then weighed, resuspended in 2:1 CHCl3:CH3OH (v/v), and stored at −20 °C until use.

Analytical and preparative thin layer chromatography

Lipids were spotted onto aluminum-backed silica thin layer chromatography (TLC) plates (EMD Chemicals Inc.) and dried. Once dry, plates were run in a solvent system with CHCl3:CH3COOH:CH3OH:H2O at a ratio of 40:25:3:6 (v/v/v/v) designed for the optimal separation of phospholipids. Plates were then dried, and sprayed with one of four TLC stains [Dittmer-Lester reagent, α-naphthol, molybdophosphoric acid (MPA), and ninhydrin] [15]. Other than Dittmer-Lester stained plates, plates were then developed by charring. For preparative TLCs, lipids were spotted across the origin of a plastic-backed TLC plate (EMD Chemicals Inc.) and were run in the same manner. After drying, a small segment of the plate was cut off for staining. The stained TLC plate section was then used to mark the lipid bands in the unstained section of the plate. The marked silica regions corresponding to CL, PG, and diglycosyldiacylglycerol (DGDG) lipid band were scraped off the plastic, moved to glass tubes, and the lipids were extracted with three sequential washes of 2:1 chloroform:methanol (C:M). After drying, lipids were resuspended in 2:1 C:M and stored at −20 °C until use.

Alkaline hydrolysis and preparation of N-(4-aminomethylphenyl) pyridinium derivative with AMPP reagent

To each fractionated PG and CL (c.a. 20 μg), and synthetic 17:0/15:0-PG, 100 μl methanol and 100 μL tetrabutylammonium hydroxide (40 wt% solution in water) were added. The solution was heated at 100 °C for 1 h. This is followed by addition of 1 mL aqueous LiCl (0.63 %) and 1 mL hexane at room temperature, vortexed for 1 min, and centrifuged at 1200×g for 2 min. The top layer containing free fatty acids was transferred to a centrifuge tube, dried under a stream of nitrogen, and N-(4-aminomethylphenyl) pyridinium (AMPP) derivative was made with the AMP+ Mass Spectrometry Kit, according to the user’s instruction. Briefly, the dried sample was resuspended in 20 μL ice-cold acetonitrile/DMF (4:1, v/v), and 20 μl of ice-cold 1 M EDCI (3-(dimethylamino)propyl)ethyl carbodiimide hydrochloride in water was added. The vial was briefly mixed on a vortex mixer and placed on ice. To the sample vial, 10 μl of 5 mM N-hydroxybenzotriazole (HOAt) 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, centrifuged for 3 min at 1200×g. The organic layer was transferred to another vial, dried under a stream of nitrogen, and stored at −20 °C until use.

Mass spectrometry

Mass spectrometric experiments with high-resolution (R = 100,000 at m/z 400), low-energy CAD, and with higher-energy collisional dissociation (HCD) were conducted on a Thermo Scientific (San Jose, CA) LTQ Orbitrap Velos mass spectrometer (MS) with Xcalibur operating system. Samples in methanol 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 100 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 35 to 70 % and with an activation q value at 0.25, and the activation time at 10 ms to leave a minimal residual abundance of precursor ion (around 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 collision-induced dissociation (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 3–10 min for MSn spectra (n = 2, 3, 4).

Results and discussion

Membrane lipids extracted from L. monocytogenes consisted of various lipid families including phosphatidylglycerol (PG) (Table 1), cardiolipin (diphosphatidylglycerol and lysylcardiolipin(lysyl diphosphatidylglycerol)) (Table 2), which is a subfamily of cardiolipin that is chemotaxonomic marker for the genus Listeria [15], and DGDG (Table 3). Interestingly, all the lipids contained the similar fatty acid substituents, mainly 12-methyltetradecanoic (anteiso-C15:0) and 14-methylhexadecanoic (anteiso-C17:0) fatty acids. The characterization of these lipid families are described below.

Table 1 Molecular species of PG from L. monocytogenes characterized by LIT MSn
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Table 2 Molecular species of CL and lysyl-CL from L. monocytogenes identified by LIT MSn
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Table 3 Molecular species of DGDG from L. monocytogenes characterized by LIT MSn
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Structural determination of phosphatidylglycerols

We previously described tandem mass spectrometric approach toward characterization of PG as [M–H]− ions. The structure assignment including the position of the fatty acid substituents on the glycerol backbone is based on the findings that the loss of the fatty acid substituent at sn-2 as an acid or as a ketene is more facile than the similar loss of the fatty acid substituent at sn-1 [23]. As shown in Fig. 1a, the LIT MS2 spectrum of the ion of m/z 721 contained the ions at m/z 497 (loss of 15:0-ketene) and 479 (loss of 15:0-acid) arising from loss of 15:0-fatty acid substituent, and the ions at m/z 469 (loss of 17:0-ketene) and 451 (loss of 17:0-acid) arising from loss of 17:0-fatty acid, indicating the presence of 15:0- and 17:0-fatty acid substituents. The ions at m/z 479 and 497 are respectively more abundant than the ions at m/z 469 and 451, indicating that the 17:0- and 15:0-fatty acid substituents are located at sn-2 and sn-1 of the glycerol backbone, respectively. The results are consistent with the observation of the ion at m/z 241 (15:0-carboxylate anion), which is more abundant than the ion of m/z 269 (17:0-carboxylate anion). The spectrum also contained the ions at m/z 647 (loss of [glycerol–H2O]), 405 (479 − [glycerol – H2O]) and 377 (451 − [glycerol–H2O]), arising from loss of the glycerol head group, giving assignment of a 17:0/15:0-PG structure [23]. Similar results were observed for the [M–H]− ion of m/z 693 (Fig. 1b), which gave rise to ions of m/z 469 (loss of 15:0-ketene) and 479 (loss of 15:0-acid) arising from losses of the 15:0-fatty acid substituents at sn-1 and at sn-2 as ketene and as acid, respectively. The ions from loss of the glycerol substituent were observed at m/z 619 (693 − [glycerol–H2O]) and 377 (451 − [glycerol–H2O]; 469 − glycerol), pointing to the presence of a 15:0/15:0-PG.

Fig. 1
figure 1

The MS2 spectra of the [M – H]− ions of 17:0/15:0-PG at m/z 721.5 (a), of 15:0/15:0-PG at m/z 693.4 (b), of p17:0/15:0-PG at m/z 705.6 (c), and its MS3 spectrum of the ion of m/z 481 (705 → 481) (d)

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Interestingly, two minor plasmalogen PG species at m/z 705 and 733 were also observed, as evidenced by high-resolution mass measurements (Table 1). The characterization of this lipid subfamily is exemplified by MSn on the ion of m/z 705.5. The MS2 spectrum (Fig. 1c), again, contained ions at m/z 481 and 463, arising from losses of the 15:0-FA acid substituent as a ketene and as an acid, respectively, along with the 15:0 carboxylate anion at m/z 241, indicating the presence of 15:0-fatty acid substituent. The spectrum also contained the ion at m/z 389, resulting from further loss of the glycerol head group. This further loss of glycerol residue is supported by MS3 on the ion of m/z 481 (705 → 481; Fig. 1d), which yielded ions at m/z 389, together with ions at m/z 227, 195 and 153 that are characteristic ions seen for PG molecules [23]. The spectrum (Fig. 1d) also contained the ion at m/z 253 (C15H31CH═CHO−), representing a heptadecenoxide anion arising from cleavage of 1-O-alk-1′-enyl residue. The confirmation of this heptadecenoxide ion was further supported high-resolution MS3 spectrum of the ion of m/z 389 (705 → 389; supplemental material Table S1), which is dominated by the ion of 253.2538 possessing an elemental composition of C17H33O (calculated, 253.2537) from loss of (glycerol–H2O) along with ion of m/z 134.9859 (elemental composition, C3H4O4P) from loss of a C15H31CH═CHOH residue. The combined structural information led to assignment of a p17:0/15:0-PG structure [24].

Structural determination of cardiolipins and lysyl-CLs

Both tandem quadrupole and LIT MSn mass spectrometric approaches have been utilized for characterization of CLs, permitting identification of the fatty acid substituents including the position of double bond and their location on the glycerol backbone [25–28]. In this study, it is noticeable that under the same ionization condition with the presence of 0.1 % NH3, only singly-charged ([M–H]−) ion was observed for the lysyl-CL (Electronic Supplementary Material Fig. S1), while doubly charged ([M–2H]2−) ions are the major ion species observed for CLs bearing no lysine residue. This is likely attributable to the fact that lysine is a basic amino acid that may form a zwitterion with the anionic phosphate group, thus deterring the further deprotonation process that yields the [M–2H]2− ions. The structural characterization conducted on the [M–H]− ions is described below.

As shown in Fig. 2a, the MS2 spectrum of the [M–H]− ion of m/z 1323 is dominated by the ions at m/z 647 (a ion) and 619 (b), together with the ions at m/z 675 (a + 56) and 703 (b + 56) and at m/z 755 (a + 136) and 783 (b + 136), indicating that the molecule consists of both a C32:0- and a C30:0-phosphatidyl moieties [25]. The MS3 spectra of the ions of m/z 647 (1323 → 647; Fig. 2b) and 619 (1323 → 619; Fig. 2c) are identical to those arising from the 17:0/15:0-PA and 15:0/15:0-PA (data not shown), respectively, pointing to the presence of 17:0/15:0- and 15:0/15:0-phosphatidyl moieties attached to the 1′ and 3′ position of the central glycerol of CL, respectively [25]. The results indicate that the ion of m/z 1323.9 represents a (15:0/15:0)(17:0/15:0)-CL, consistent with the observation of the ions at m/z 423 (a − 15:0-ketene), 405 (a − 15:0-FA), 395 ([b − 15:0-ketene] + [a − 17:0-ketene]), and 377 ([b − 15:0-FA] + [a − 17:0-FA]) in Fig. 2a. The structural assignments are further supported by the accurate mass measurement of the individual ions by high-resolution MS2 (ESM Table S2).

Fig. 2
figure 2

The MS2 spectrum of the [M – H]− ions of (17:0/15:0)(15:0/15:0)-CL at m/z 1323.9 (a), and its MS3 spectra of the ion of m/z 647 (1323 → 647) (b), of the ion of m/z 619 (c). The ion nomenclature in Fig. 5a is adopted from ref. [25]

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By contrast, the MS2 spectrum of the [M–H]− ion of m/z 1451 (Fig. 3a) (Table 2) is dominated by the ions at m/z 1323 (loss of [lysine − H2O]) and 1305 (loss of lysine), arising from elimination of the lysine residue, along with the ions at 647 (a ion) and 619 (b), respectively representing a C32:0- and a C30:0-phosphatidyl substituents as seen earlier (Scheme 1a). The MS3 spectrum of the ion of m/z 1323 (1451 → 1323; data not shown) is identical to the MS2 spectrum of the [M–H]− ion of m/z 1323 shown in Fig. 2a. The results led to the assignment of lysyl-(15:0/15:0)(17:0/15:0)-CL.

Fig. 3
figure 3

The MS2 spectrum of the [M – H]− ions of lysyl-(17:0/15:0)(15:0/15:0)-CL at m/z 1452.0 (a), and its MS3 spectra of the ion of m/z 1249.8 (1452 → 1249) (b), and its MS4 spectrum of the ion of m/z 709 (1452 → 1249 → 709) (c). The ion of m/z 1249 arises from internal loss of a lysylglycerol residue which is unique to lysyl-CL (the proposed fragmentation pathways are shown in Scheme 1)

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Scheme 1
scheme 1

The fragmentation processes proposed for lysyl-CL. The numbers reflect the masses (m/z) of the fragment ions observed for the [M - H]− ion of (17:0/15:0)(15:0/15:0)-lysyl-CL

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In Fig. 3a, an ion at m/z 1249 was also observed. High-resolution mass measurement gave an elemental composition of C68H131O15P2 (calculated, 1249.8969; measured, 1249.8951) (see supplemental material Table S3) reflecting that the ion may arise from internal expulsion of a lysylglycerol residue (loss of C9H18O3N2), to form a diphosphatidyl anion (Scheme 1b). This speculation is further supported by the MS3 spectrum of the ion of m/z 1249 (1451 → 1249; Fig. 3b), which contained the ions at m/z 1025 (loss of 15:0-ketene) and 1007 (loss of 15:0-acid) arising from loss of 15:0-fatty acid residue, and ions at m/z 979 (loss of 17:0-ketene) and 953 (loss of 17:0-acid) arising from elimination of 17:0-fatty acid residue, consistent with the presence of 15:0- and 17:0-fatty acyl groups in the molecule. The spectrum also contained abundant ions at m/z 709 and 681, arising from losses of the 15:0/15:0- and 17:0/15:0-diacylglycerol residues, respectively (Scheme 1b). Further dissociation of the ion of m/z 709 (1451 → 1249 → 709; Fig. 3c) yielded 485/467 and 457/439 ion pairs arising from cleavages of 15:0-, and 17:0-fatty acid substituents as ketene and as acid, respectively, along with ion at m/z 215 arising from further loss of the remaining fatty acid substituent (Scheme 1b). The results are consistent with the diphosphatidyl structure suggested for the ion of m/z 1249, confirming the fragmentation process of the internal residue loss. In contrast, an internal loss of a corresponding glycerol residue was not observed for the cardiolipins that do not bear the lysine residue (e.g., Fig. 2a).

Characterization of diglycosyldiacylglycerol

In the positive ion mode, several diglycosyldiacylglycerol (DGDG) species in the [M + Na]+ form were observed and the major constituent was seen at m/z 915 (Table 3). The MS2 spectrum (Fig. 4a) contained ions at m/z 753 arising from loss of a hexose residue, together with abundant ions at m/z 645 and 673 arising from losses of 17:0- and 15:0-fatty acid substituents, respectively. The ion at m/z 645 is more abundant than the ion at m/z 673, indicating the 17:0-fatty acyl group is located at sn-1; while the 15:0-fatty acyl group is located at sn-2 of the glycerol backbone. The spectrum also contained the minor ions at m/z 483 and 511 arising from combined losses of the hexose residue plus the 17:0- and 15:0-fatty acid substituents, respectively. The structural assignments were further confirmed by the MS2 spectrum of the corresponding [M + Li]+ ions at m/z 899 (Fig. 4b), in which a mass shift of 16 Da was observed for all the fragment ions. The results gave assignment of a 17:0/15:0-DGDG structure [29, 30].

Fig. 4
figure 4

The MS2 spectra of the [M + Na]+ ions of 17:0/15:0-DGDG at m/z 915.6 (a), and of the corresponding [M + Li]+ ions at m/z 899.7 (b). The losses of the fatty acid substituents in Panel a are confirmed by the mass shift of the corresponding ions in Panel b

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Characterization of the anteiso-C15:0 and anteiso-C17:0 fatty acid substituents in PG, DPG, and DGDG

The AMPP derivatives of the FA substituents arising from the fractionated CL, and PG, after alkaline hydrolysis all gave rise to the major M+ at m/z 437 and 409 (data not shown). HCD MS2 spectrum of the ions at m/z 437 (Fig. 5a) is identical to that of the anteiso-C17:0-AMPP standard (not shown). The spectrum contained abundant ions at m/z 169 and 183, along with m/z 211 that are characteristic fragment ions seen for the fatty acid-AMPP derivatives [18–21]. Figure 5a is featured by the ion series of m/z 239, 253, 267, 281, 295, 309, 323, 337, 351, 365, and 379, arising from cleavages of the straight of C–C bond, along with ion of m/z 407 arising from removal of the terminal ethyl group (loss as CH2═CH2 + H2), supporting that the methyl side chain is located at the n−3 of the hydrocarbon chain and the presence of the anteiso-C17:0 fatty acid structure. The low-energy CAD MS2 spectrum of the ion of m/z 437 is similar to that shown in Fig. 5a (data not shown), but the heavier ions of m/z 365, 379, and 407 that are crucial for locating the methyl side chain are less abundant, compromising its utility in the confident assignment of the methyl group, although applicable.

Fig. 5
figure 5

The HCD MS2 spectra of the [M + H]+ ions of the anteiso-17:0-AMPP derivative at m/z 437 (a), and of anteiso-15:0-AMPP derivative at m/z 409 (b) from L. monocytogenes, and of the standard iso-17:0-AMPP derivative at m/z 437 (c). The location of the methyl side chain is located by the observation of the 28 Da gap as shown in the fragmentation schemes in the inset of each spectrum

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Similar fragment ions were also observed in Fig. 5b, however, the spectrum is featured by the ion series of m/z 239, 253, 267, 281, 295, 309, 323, 337, and 351 together with 379, indicating that the C15:0 represents a anteiso-C15:0 structure. In contrast, the HCD MS2 spectrum of the M+ ion of the standard iso-C17:0-AMPP isomer at m/z 437 contained the ion series of m/z 239, 253, 267, 281, 295, 309, 323, 337, 351, 365, 379, and 393, arising from cleavage of the C–C bond of the straight chain, along with ion of m/z 421 arising from removal of the terminal methyl group as methane, pointing to an iso-C17:0 structure (Fig. 5c).

Conclusions

We applied LIT MSn with high-resolution mass spectrometry in combination of chemical reactions toward complete structural identification of polar lipids isolated from membrane of L. monocytogenes. It is interesting to note that anteiso-C15:0 and anteiso-C17:0 fatty acids are the major fatty acid substituents found in all the lipid families analyzed; and the anteiso-C17:0 and anteiso-C15:0 are mainly located at sn-1 and sn-2 of the glycerol backbone, respectively. The structure similarities among PG, CL, and DGDG are also consistent with the biosynthetic pathways of CL [31] and DGDG [32–34] in the bacteria. The observation of the rare plasmalogen PGs that have not been previously reported in Listeria is interesting; and the biological significance underlying the formation may warrant further studies.