Liquid Chromatography with Electrospray Ionization Tandem Mass

Chapter 4

Liquid Chromatography with Electrospray Ionization Tandem Mass Spectrometry Profiling Carbohydrates in Whole Bacterial Cell Hydrolysates Downloaded by KTH ROYAL INST OF TECHNOLOGY on November 21, 2015 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch004

1

Gavin E. Black and Alvin Fox

Department of Microbiology and Immunology, School of Medicine, University of South Carolina, Columbia, SC 29208 Carbohydrates can serve as chemical markers that allow for the taxonomic differentiation of bacteria. Gas chromatography-mass spectrometry (GC-MS) is a proven technique for profiling neutral and amino sugars(asalditolacetates)in bacterial cell hydrolysates. The chromatograms display low background and mass spectra are readily interpretable. Unfortunately, the required derivatization is time consuming and not applicable to all sugars of interest. This review concerns the profiling of underivatized sugars using electrospray ionization tandem mass spectrometry with either direct injection (MSMS) or on-line liquid clrornatography (LC-MS-MS). Both MS-MS and LC-MS-MS can facilitate the rapid identification of carbohydrates over conventional GC-MS. However, unlike MS-MS, LC-MS-MS can readily discriminate sugar isomers. Sugar standards can be chromatographically resolved and thus be analyzed by the rather non-specific pulsed amperometric detector. However, chromatograms obtained for bacterial whole cell hydrolysates are complicated and require the specificity of MS for analysis. For MS analysis, following high pH anion exchange chromatography, a cation suppressor is used to remove sodium hydroxide from the eluent. Amino sugars are removed in the suppressor. Thus analyses are focussed on profiling of acidic and neutral sugars. The LC mobile phase generates considerable background ions on MS analysis, thus MS-MS is vital to obtain product ion spectra for sugar identification. LC-MS-MS instrumentation is more expensive than GC-MS and profiles different groups of sugars. However, sample preparation for LC-MS-MS and MS-MS is simpler in comparison to GC-MS. Electrospray (ES) is a well established technique allowing mass spectrometry (MS) analysis of polar compounds from aqueous solution without derivatization. For this reason, ES is exquisitely suited for on-line analysis in conjunction with liquid chromatography (LC). The potential of LC-MS for analysis of sugar monomers has been 1

Corresponding author 0097-6156/95/0619-0081$13.25/0 © 1996 American Chemical Society

In Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry; Snyder, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

Downloaded by KTH ROYAL INST OF TECHNOLOGY on November 21, 2015 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch004

82

BIOLOGICAL AND BIOTECHNOLOGICAL APPLICATIONS OF ESI-MS

demonstrated (1-3), however their analysis in more complex matrices requires further investigation. High pH anion exchange LC (HPAEC) with pulsed amperometric detection (PAD) is suitable for profiling of sugar monomers (4). Due to the simplicity and sensitivity of LC-PAD it has rapidly become one of the most common techniques for chromatographic analysis of carbohydrates. However, PAD is non-selective and is unsuitable for complex biological matrices (5, 6). For complex samples, the specificity offered by MS is essential. Microbial identification and taxonomic differentiation are traditionally accomplished by culture-based methods to determine physiological characteristics. Alternatively, identification can be achieved by determining the presence of unique sugars (chemical markers) as alditol acetate derivatives utilizing GC-MS analysis (7-9). The alditol acetate procedure allows derivatization of neutral and amino sugars but not acidic sugars. This limitation arisesfromthefeetthat hydroxyl and amino groups are amenable to acylation but carboxylic acid functions are not. There is considerable diversity of carbohydrates among bacterial species, many of which are readily identifiable by GC-MS. Sugars exist in a multitude of stereoisomers. These stereoisomers cannot be readily distinguished on grounds of their molecular mass but often have different chromatographic retention times (10). Thus chromatographic characteristics are extremely important in sugar identification. An excellent review detailing all known monosaccharide components of bacterial polysaccharides (85 in total) reported in the literature before 1989 has been published. As noted by Lindberg "only a limited number of all the bacterial families and tribes have been investigated for their cell wall or extracellular polysaccharides or both" (11). A brief overview of some aspects of sugar chemistry provides a perspective on variability in sugar structure. Further information on sugar structure is provided elsewhere (12). Sugars are polyhydroxyl compounds and their backbones commonly containfrom3-7 carbons. Each internal carbon acts as an optical center. For example, for a simple hexose (e.g. glucose) there are 4 optical centers or 4 isomers. Monosaccharides commonly exist in both aldose and ketose forms (carbonyl function in either position 1 or 2 respectively). Replacement of hydroxyl and/or aldehyde functions with amino or carboxylic acid functions leads to aminosugars, alduronic acids (COOH in position 6), aldonic acids (COOH in position 1) and aldaric acids (COOH in position 1 and 6). The primary and secondary hydroxyl groups may be reduced to give methyl or deoxysugars respectively and dehydration of acidic or neutral sugars leads to lactone and anhydro sugars respectively. Derivatization of the functional groups (acylation, methylation) is also commonly observed. As an example of taxonomic discrimination provided by carbohydrate markers, muramic acid (MA, (3-O-lactyl glucosamine) is a sugar common to almost all eubacteria (as a component of cell wall peptidoglycan) while not present in non-bacterial matter, including fungi (13,14). In a wider sense, neutral and aminosugar profiles allow discrimination among Gram negative bacteria and Gram positive bacteria. For example, aminodideoxyhexoses are useful for distinguishing among the Legionellaceae. Fucosamine is characteristic of the genus Legionella whilst quinovosamine is found in the genus Tatlockia (15,16). O-methylated sugars are useful in discriminating various Bacillus species. Thus 2-O-methyl and 3-O-methyl-rhamnose are found in the spores of B. cereus but only the 3-O-methyl isomer in B. anthracis (17) . 2



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Liquid Chromatography with ESI-MS

83

Profiling of carbohydrates by GC-MS of the corresponding alditol acetates has become routine in our laboratory. It is noteworthy that chemotaxonomic speciation provided by carbohydrate profiles agrees closely with molecular differentiation techniques such asribosomalRNA sequencing (18) and PCR amplification of ribosomal RNA spacer regions (19). Unfortunately, GC-MS analysis requires derivatization which is both labor intensive and time consuming. Simple and rapid analysis is achievable by ES-MS-MS without prior chromatography. However, suppressionfromsample matrix components can adversely affect detection limits (20). Furthermore, much chemotaxonomic information resides in the differentiation of sugar isomers (15-19). Such isomers are unlikely to be readily discriminated by MS-MS analysis. On the other hand, sugar isomers can be distinguished on the grounds of different chromatographic retention times (16,17). For example, B. anthracis and B. cereus can often be distinguished by the presence of galactose although both species commonly contain glucose (17). Due to interferences, unambiguous identification of sugars in complex matrices requires high detection specificity. The latter may be provided by LC-MS, or better yet, LC-MS-MS. Because LC-MS-MS does not require derivatization it offers an appealing alternative or complementary technique to GC-MS profiling Excellent chromatography of native sugars can be obtained using HPAEC with pulsed amperometric detection (PAD) (21). However, PAD is not a selective detector, (5, 6) and for detection of sugars in complex matrices (e.g. bacterial whole cell hydrolysates) MS is required. Unfortunately, the high ionic strength solutions necessary for this type of LC separation are not well suited to MS (2,3). This limitation may be overcome by using on-line suppressors (replacing NaOH with H 0) but the price paid for it is that positively charged chemotaxonomic markers, such as aminosugars, are lost in the process. The purpose of this review is to describe the current status of carbohydrate profiling by LC-ES-MS, ES-MS-MS and LC-ES-MS-MS. Reviews on the current status of GC-MS can be found elsewhere (7-9). 2

Direct injection mass spectrometry and tandem mass spectrometry Electrospray ionization followed by MS-MS analysis has been used for the analysis of underivatized carbohydrates from whole cell hydrolysates (20). MA wasfirstreleased by hydrolysis in 2N sulfuric acid. Acid was then removed by extraction with N N dioctylmethylamine in chloroform followed by hydrophobic clean-up using C-18 columns. No derivatization was performed and sample processing time was reduced to ~4 hr compared to a 50 hr preparation time for GC-MS. Additionally, the elimination of chromatography reduced instrumental analysis timefrom60 min to 2 min per sample. For compounds present at low levels, however, signal suppression by components of the sample matrix adversely affected sensitivity, often making product ion spectra unobservable. For aminosugar analysis, positive ion ES produced abundant molecular ions (M+H) with little fragmentation. Acidic sugar analysis, using negative ion ES, produced primarily deprotonated molecular ions (M-H)". Figure 1 shows ES MS spectra of f

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glucuronic acid (GlcUA, mol.wt. 194) and 3-deoxy-D-manno-octulosonic acid (KDO, molecular wight 238) as examples of acidic sugars. These sugars both form predominantly deprotonated molecular ions (m/z 193 for GlcUA, m/z 237 for KDO). Neutral sugars ionize weakly under both positive ion and negative ion ES conditions. However, neutral sugars can form adducts under both positive and negative ion conditions. Neutral sugars, including glucose and fucose, have been detected using positive ion ES as their Na , N H and L i adducts (2,3). It has been observed for plasmaspray (2) and thermospray (3) analysis, that formation of ammonium adducts were necessary for sugar ionization. The negative ion mode has been used for detection of low levels of anhydroglucitol in serum in form of chlorine adduct ions (22). In the presence of acetate, neutral sugars generate [M+AcO] 'and [M-H] " under negative ion ES-LC-MS conditions (unpublished data). For compounds containing both carboxyl and amino groups, such as phospholipids (23) mycotoxins (24) and MA positive and negative ionization can be used to increase selectivity. Additional specificity is provided by collision-induced dissociation (CID). For example, in the positive ion mode, MA produced a prominent protonated molecular ion at m/z 252 [M+H] (20) whereas in the negative ion mode the deprotonated ion 250 [M-H]" was observed (9). The precursor ion m/z 250 generated an ion at m/z 89, presumablyfromthe loss of a lactate anion on CID (9). MA is known to lose lactic acid under alkaline conditions and this fact has become the basis for a spectrometric assay for MA (25,26). Multiple cellular components in whole cell hydrolysates produce a complex mixture of signals. Thus full scan or selected ion monitoring does not allow definitive detection of chemical markers. When chemical markers are present at high concentrations, it is possible to obtain product ion spectra. However, the increased sensitivity of multiple reaction monitoring (MRM) is often essential. In this instance, a characteristic ion (selected in thefirstmass analyzer) produces specificfragmentsafter CID, which are monitored in the second mass analyzer. MS-MS produces lower ion intensities than MS analysis, due to poor transmission between the two mass analyzers, but improved signal to noise ratio leads to lower detection limits. As an example of MS-MS analysis of whole cell hydrolysates, monitoring for ions characteristic of MA did not allow discrimination of bacteria and fungi when ES-MS was used. However, monitoring for product ions of MA allowed ready differentiation of bacteria and fungi (9,20). The acidic sugar, KDO, is present in the lipopolysaccharides (LPS) of most, but not all, Gram negative bacteria. MS-MS analysis of authentic KDO produced an identicalfragmentspectrum to KDO from a hydrolysate of the Gram negative bacterium Legionella pneumophila (see Figure 2). When complex samples containing multiple compounds enter the ES source, ion suppression is a commonly observed phenomenon with some compounds ionizing better than others. Thus, despite the sensitivity of ES, the limit of detection may be greatly diminished as compared to pure compounds. For example, GlcUA is present in the hyaluronic acid capsules of certain group A and group C streptococci (27), we were unable to detect GlcUA in streptococcal hydrolysates using ES-MS-MS with direct injection. It required on-line LC-MS to detect this sugar (see following text). +

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Liquid chromatography-mass spectrometry Many LC systems for sugar analysis have been described but few provide both high resolution separations and column durability (28, 29). One column that displays these characteristics is an HPAEC column (PA-1) developed by Dionex. This column has been primarily used in conjunction with a PAD detector (4,21,28). As noted above, it may be of advantage to combine HPAEC with ES-MS or ES-MS-MS. However this combination is not straightforward (2,3). To ionize carbohydrates for HPAEC, high concentrations of NaOH (18-100 mM) are necessary. For acidic sugars (including KDO and GlcUA), a gradient of Na AcO" was needed for displacement from the anion exchange resin (28). A cation suppressor exchanges the sodium ions in solution with hydrogen ions, thus replacing sodium hydroxide with water. A 2 mm anion self-regenerating suppressor from Dionex (Sunnyvale, CA) was used in our studies. This anode-based electrolysis system produces H and 0 fromH 0. The generated H ions replace Na (present in the mobile phase in the form of Na AcO"). Removal of Na thus generates acetic acid, which is tolerated by ES ionization. Using a 2 mm column, the flow rate can be reduced to 100 μΐ/min. Whilst not entirely optimal for chromatographic resolution thisflowrate of concentrated NaOH is readily handled by the ion suppression system. The potential of HPAEC LC-MS, was demonstrated previously using thermospray (2) and ES (3) for the analysis of sugar standards. Amino-containing compounds (eg. aminosugars) do not pass through the suppressor. In complex samples which contain a large amount of protein, high concentrations of amino acids are generated. The presence of the on-line suppressor effectively removes these amino acids, thus acting as an on-line clean-up procedure. Previous LC-MS studies have not addressed the analysis of acidic sugarsfrombacterial cell hydrolysates. As mentioned above, acidic sugars are particularly difficult to analyze by GC-MS due to their carboxyl groups which require additional derivatization. Certain alternative columns can be used at neutral pH (eliminating the need for an ion suppressor) which makes them more compatible with MS detection, but chromatographic resolution can be compromised. Previous LC-MS studies have relied upon post-column addition of reagents that promote adduct ion formation (3,22). Instead the desired adduct ions may also be formed without the need of post-column reagents by selecting an appropriate LC buffer. For example, we have used Na AcO" gradients to form the molecular acetate adduct ions (unpublished). Deoxyhexoses (dHex), including rhamnose and rucose, have a molecular weight of 164 and under negative ion ES these sugars formed both acetate adduct ions ([dHex+AcO]' = m/z 223) and deprotonated molecular ions ([dHex-H]" = m/z 163). Similarly glucose (mol.wt. 180) and ribose (mol.wt. 150), both commonly found in bacteria, produced acetate adduct ions [M+AcO]' ; m/z 239 and m/z 209 respectively and deprotonated molecular ions, [M-H]" ; at m/z 179 and m/z 149 respectively. Sometimes dimers are observed e.g. [Hex-Hex]" at m/z 359. Acidic compounds formed primarily deprotonated molecular ions; gluconic acid (GlcOA, used as an internal standard) which has a molecular weight of 196 formed an ion of m/z 195, N-acetyl neuraminic acid (mol. wt. 309) observed at m/z 308, KDO (mol. wt. 238) at m/z 237 and glucuronic acid (GlcUA, mol. wt. 194) at m/z 193. +

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Figure 2. Product ion spectra of the [M-H]" obtained by ES-MS-CID-MS. (A) glucuronic acid standard, (B) authentic KDO, and (C) KDO from the hydrolysate of Legionella pneumophila. The spectra for authentic KDO and KDO isolated from bacteria are essentially identical.

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In total ion chromatograms, peaks characteristic of sugars were observed on a high baseline or background signal generated by components of the mobile phase (e.g. AcO", m/z 59). In the SIR mode data acquisition was focussed only on the deprotonated molecular ion (for neutral and acidic sugars) or acetate adduct ions (neutral sugars) rather than full scans. Chromatograms showed signals for all the compounds in the standard mixture (compare Figures 3 and 4) except for the amino sugars. For example, as seen in the LC-MS SIR chromatogram monitoring for m/z 250 [Μ-Η'] showed the absence of MA although a peak for this compound is readily seen in the LC-PAD chromatogram. As noted above, amino-containing compounds are unable to pass through the cation permeable suppressor. Bacterial hydrolysates presented considerably more complicated LC-PAD chromatograms (Figure 5) than standards (Figure 3). Even when set to optimize sugar detection, the PAD is not a specific detector. There are many compounds present in high concentrations in the bacterial cell which may contribute significant background response. However, LC-MS SIR chromatograms allowed ready identification of neutral and acidic sugars in whole cell hydrolysates. For example, as shown in Figure 6, rhamnose, glucose,riboseand GlcUA acid were readily detected in S. zooepidemicus. GC-MS analysis of alditol acetates of this organism confirmed the presence of these neutral sugars. Additionally GC-MS analysis showed the presence of aminosugars including MA, glucosamine, and galactosamine (data not shown). Thus LC-MS and GCMS together produce complementary sugar profiles allowing identification of amino, neutral, and acidic sugars. Carbohydrates were also generated by hydrolysis of whole cells of the Gram negative organism, Legionella pneumophila. LC-MS demonstrated the presence of rhamnose, hexoses,ribose,and KDO (see Figure 7). Under these chromatographic conditions mannose and glucose were not resolved. Previous GC-MS studies showed the presence of the neutral sugars rhamnose, mannose, glucose, andribose,in addition to the aminosugars MA, glucosamine and quinovosamine (16). Similarly, GC-MS has previously demonstrated the presence of KDO in L. pneumophila (32). To determine the sensitivity of this technique, spiking experiments were performed. GlcUA acid (not present in L. pneumophila) corresponding to 0.01 through 10.0% of the bacterial dry weight in each sample was added to cell hydrolysates. The lowest level of detection, using SIR, corresponded to 0.05% (data not shown).

Liquid chromatography-tandem mass spectrometry The common ionization techniques for GC-MS are electron ionization (EI) and chemical ionization (CI). The former is used for structural identification and produces fragment mass spectra, whereas CI produces intact molecular ions and is helpful in establishing molecular weight. As noted above, ES produces primarily intact, molecular ions, althoughfragmentationcan be accomplished by varying ionization conditions. In the positive ion mode aminosugars gain a proton, whilst in negative ion mode acidic sugars lose a proton. However, due to the high abundance of low mass ionsfromthe LC eluent, in-source production offragmentionsfromthe small molecules can be difficult



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Retention Time (min) Figure 7 - SIR chromatograms of a L pneumophila hydrolysate. Peak identifications are the same as in Figure 3. The numbers on therighthand side of each chromatogram are the same compounds listed in Figure 3. The numbers above each peak refer to the m/z ratios monitored at the respective retention time. Continued on next page

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to distinguishfrombackground. Instead, MS-MS allowed selective transmission of the highest abundance molecular (or adduct) ion followed by generation of product ion spectra which were substantially free from background noise. Detection of the molecular ion in thefirstmass analyzer in conjunction with product ions in the second analyzer is analogous to performing separate CI and EI analyses for GC-MS. The neutral sugar ribose is present at high levels in bacterial cells, principally as a component in nucleic acids. Figure 8 compares product ion spectra obtained by LCMS-MS of authenticriboseandribosepresent in a hydrolysate of L pneumophila. Both product ion spectra of m/z 209 [M-AcO]" contained m/z 149, m/z 89, m/z 71, and m/z 59 (see Figure 8). Acidic compounds did not form adducts but formed exclusively their deprotonated molecular ions. Product ion spectra of KDO in a hydrolysate of L pneumophila were also identical tofragmentsproduced by a KDO standard (Figure 9).

Current status of LC-MS-MS, MS-MS and GC-MS for profiling sugars in bacterial hydrolysates

Sample preparation for LC-MS-MS or MS-MS analysis of carbohydrates in whole cell hydrolysates requires no derivatization. The entire procedure consists of three steps (acid hydrolysis, removal of the acid, and solid phase extraction with C-18 columns) and takes a few hours (20). In contrast, sample preparation for GC-MS analysis of bacterial carbohydrates is a multi-step procedure which currently requires three days (7-9). Sugar analysis using HPAEC-LC is performed using concentrated sodium hydroxide in the mobile phase. Standard mass spectrometers are not designed to handle high concentrations of NaOH present in the LC eluent. Thus a cation permeable ion suppressor is placed after the LC column and prior to the MS. The ion suppressor exchanges hydrogen for sodium ions; thus replacing sodium hydroxide with water. Other cationic species (including basic and amphoteric compounds) are removed by the suppressor and this serves as an on-line clean-up step (e.g. in removing amino acids and peptides) (2,3). In GC-MS analysis, total ion chromatograms of neutral and aminosugars (as alditol acetate derivatives)frombacterial cell hydrolysates display low background in the total ion chromatogram. Full spectra also display low background and are readily interpreted. In LC-MS analysis, there is considerable generation of signal from the components of the mobile phase. By selection of a parent ion in the first mass analyzer, product ion mass spectra free of contribution from the mobile phase are observed. However, LC-MS-MS (triple quadropole) instrumentation is considerably more expensive than GC-MS instruments. Commercial benchtop GC-MS instruments have been available since the 1980's whereas user-friendly LC-MS and LC-MS-MS instruments have only become available in the 1990's. Further development of LC-MS-MS instruments for routine use (including simplification of software and further integration of the control of LC, MS-MS and autosamplers) is vital. The recent availability of modestly priced ion trap instruments that can be operated in the LC-MS-MS mode may contribute to rapid changes in both



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Liquid Chromatography with Electrospray Ionization Tandem Mass

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