Linking Mass Spectrometry and Slab-Polyacrylamide Gel

Haemophilus influenzae is an important cause of hu- man disease, and its lipopolysaccharide (LPS) is known to be a major virulence factor. H. influenz...
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Anal. Chem. 2003, 75, 4918-4924

Linking Mass Spectrometry and Slab-Polyacrylamide Gel Electrophoresis by Passive Elution of Lipopolysaccharides from Reverse-Stained Gels: Analysis of Gel-Purified Lipopolysaccharides from Haemophilus influenzae Strain Rd Sofia Gulin,† Elder Pupo,‡ Elke K. H. Schweda,† and Eugenio Hardy*,‡

Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, NOVUM, S-141 86 Huddinge, Sweden, and Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba

Haemophilus influenzae is an important cause of human disease, and its lipopolysaccharide (LPS) is known to be a major virulence factor. H. influenzae produces short-chain LPS of which the heterogeneity is often visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using silver staining for detection. Individual bands have not previously been recovered by this method in quantities sufficient for mass spectrometry. In an attempt toward the development of sensitive mass spectrometrical strategies to be used in structural studies of H. influenzae LPS and LPS from other bacteria, we have applied here our previously described slab-PAGE-based micropurification method to obtain unmodified LPS fractions of high purity (>95%) from a crude LPS preparation of H. influenzae strain Rd. Two LPS-fractions were obtained which, after a procedure including mild acid hydrolysis, dephosphorylation, and permethylation of the resulting oligosaccharides, were subjected to tandem electrospray ionization mass spectrometry (ESI-MS/MS). The quantities of micropurified LPS fractionssthe recovery of LPS in terms of total mass was 30%swere found sufficient to allow the characterization of LPS glycoforms. The ESI-MS spectra of the individual bands showed reduced heterogeneity. Furthermore, the integrity of the micropurified LPS was confirmed. The spectra-displayed molecular ions showed improved intensity, increased respective signal-to-noise ratios demonstrating the sensitivity of analysis. Consequently, both the direct determination of the molecular masses of the gel-separated LPS glycoforms and sequence analyses using ESI-MS/MS were possible.

* To whom correspondence should be addressed. Fax: 537.33 6008, 537.271 8070, or 537.271 8675. E-mail: [email protected]. † Clinical Research Centre, Karolinska Institutet and University College of South Stockholm. ‡ Center for Genetic Engineering and Biotechnology.

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Glycoconjugates and proteins located in exposed positions on the outer cell membrane of pathogenic gram-negative bacteria are involved in important interactions between the bacteria and the host organism. Lipopolysaccharide (LPS) as a prominent outermembrane structural feature shows essential physiological functions for the bacteria and exhibits a variety of biological and immunological activities in higher organisms. In many bacteria LPS is composed of a largely conserved lipid-A moiety anchored in the cell membrane followed by an oligosaccharide core region and a terminal O-specific chain which is a variable polysaccharide built up of repeating units. The lipid A and core region are linked together through one or more acidic sugars identified as 3-deoxyD-manno-2-octulosonic acid (Kdo). A number of nonenteric gram-negative bacteria, including the genus Haemophilus, produce LPS that lack repeating polysaccharide structures and instead contain a more variable and branched core oligosaccharide region linked to the relatively invariant lipid A. Haemophilus influenzae is a bacterium that routinely colonizes the upper respiratory tract of humans. It is also the cause of both upper and lower respiratory tract infections that result from contiguous spread. Occasionally, H. influenzae can cause systemic and life-threatening bacteraemic diseases such as septicemia and meningitis. LPS is an essential and integral surface component of H. influenzae and functions as a major virulence determinant. Determination of structure is crucial to understanding the biology of H. influenzae LPS and its role in bacterial virulence. The complexity of LPS molecules requires sophisticated analytical techniques and often the use of several chemical methods to bring about their structural characterization. Structural analyses are usually performed on material obtained from large-scale growth of bacteria and require appreciable amount of material. A routinely used analytical separation technique of LPS is polyacrylamide gel electrophoresis (PAGE). PAGE in the presence of sodium dodecyl sulfate (SDS) or sodium deoxycholate (DOC) is a powerful tool for resolving complex mixtures of lipopolysaccharides in a wide molecular weight range. SDS- or DOC-PAGE 10.1021/ac034365o CCC: $25.00

© 2003 American Chemical Society Published on Web 08/19/2003

followed by silver staining1 allows sensitive, simple, and rapid profiling of LPS.2-7 This method provides information on the heterogeneity of LPS including the number of component glycoforms, their relative abundance, and approximate molecular masses.8,9 Further information on LPS properties has been obtained from PAGE-separated bands after their blotting on membranes and characterization by immunological or binding assays10-12 or after passive elution or electroelution and subsequent analysis by diverse in vitro and in vivo biochemical tests.13,14 Other separation techniques such as HPLC and CE are relatively easily interfaced with electrospray ionization mass spectrometry (ESI-MS), thus facilitating the simultaneous separation and determination of minute amounts of carbohydrates. Recently, thin-layer chromatography (TLC) has been combined with MALDI mass spectrometry for the direct microextraction and improved analysis of native rough-type LPS or lipid A.15 The analysis of slab PAGE separated LPS by mass spectrometry, however, has been hampered because of two main reasons. First, due to both the amphiphilic and amphiphatic nature of LPS, it is not easily recovered in its native form from polyacrylamide gels by most commonly used elution methods such as passive diffusion or electroelution. In fact, in the absence of SDS most of the gel-separated LPS remain in the gel matrix even after longterm incubation in water or electroelution.16 In addition, conversion of LPS-band-containing gel slices into gel microparticles of 32µm average size, which has previously shown to allow quantitative passive diffusion of water-soluble proteins and nucleic acids from polyacrylamide gels into water,17-19 only provides an improved, yet variable recovery of low-molecular-weight LPS.20 Accordingly, LPS could originally be recovered from polyacrylamide gels only in the presence of LPS deaggregating agents (e.g., 0.1% SDS), although with a modest efficiency (37.5%) and just after a prolonged time of electroelution (5 h or overnight).14 Second, since conventional and most sensitive methods for visualizing gel(1) Tsai, C. M.; Frasch, C. E. Anal. Biochem. 1982, 119, 115-119. (2) Lawson, A. J.; Chart, H.; Dassama, M. U.; Threlfall, E. J. Lett. Appl. Microbiol. 2002, 34, 428-432. (3) Zhu, P.; Klutch, M. J.; Tsai, C. M. FEMS Microbiol. Lett. 2001, 203, 173177. (4) Lee, B. J.; Hampson, D. J. J. Med. Microbiol. 1999, 48, 411-415. (5) Jurgens, D.; Fehrenbach, F. J. J. Clin. Microbiol. 1997, 35, 3054-3057. (6) Guard-Petter, J.; Lakshmi, B.; Carlson, R.; Ingram, K. Appl. Environ. Microbiol. 1995, 61, 2845-2851. (7) Aucken, H. M.; Pitt, T. L. J. Clin. Microbiol. 1993, 31, 1286-1289. (8) Komuro, T.; Nakazawa, R. Int. J. Artif. Organs 1993, 16, 245-248. (9) Amano, K.; Mizushiri, S.; Fukushi, K. Nippon Saikingaku Zasshi 1988, 43, 911-916. (10) Tsai, C.; Chen, W.; Balakonis, P. Glycoconjugate J. 1995, 12, 562. (11) Inzana, T. J.; Glindemann, G.; Cox, A. D.; Wakarchuk, W.; Howard, M. D. Infect. Immun. 2002, 70, 4870-4879. (12) Brade, L.; Podschun, R.; Brade, H. J. Endotoxin Res. 2001, 7, 119-124. (13) Sandbulte, J.; TerWee, J.; Wigington, K.; Sabara, M. Vet. Microbiol. 1996, 48, 269-282. (14) Ohta, M.; Rothmann, J.; Kovats, E.; Phan, P. H.; Nowotny, A. Microbiol. Immunol. 1985, 29, 1-12. (15) Therisod, H.; Labas, V.; Caroff, M. Anal. Chem. 2001, 73, 3804-3807. (16) Hardy, E.; Pupo, E.; Castellanos-Serra, L.; Reyes, J.; Fernandez-Patron, C. Anal. Biochem. 1997, 244, 28-32. (17) Castellanos-Serra, L. R.; Fernandez-Patron, C.; Hardy, E.; Santana, H.; Huerta, V. J. Protein Chem. 1997, 16, 415-419. (18) Castellanos-Serra, L. R.; Fernandez-Patron, C.; Hardy, E.; Huerta, V. Electrophoresis 1996, 17, 1564-1572. (19) Castellanos-Serra, L. R.; Hardy, E.; Sa´nchez, J. C. Anal. Biochem. 1998, 257, 227-228. (20) Hardy, E.; Pupo, E.; Santana, H.; Guerra, M.; Castellanos-Serra, L. R. Anal. Biochem. 1998, 259, 162-165.

separated LPSssilver staining and more recently the fluorescent dye-LPS conjugate detection methods1,21cause chemical modification and irreversible fixation of these macromolecules in the gel, the recovery of separated LPS bands for subsequent biochemical characterization has been hindered. In the way to overcome these major obstacles, some improvements of the separation of LPS by slab-PAGE including the use of highly resolutive buffer systems,22 LPS-deaggregating detergents,23 polyacrylamide gel concentration gradients,24,25 or optimization for particular crude LPS have been described.2,26 In addition, a sensitive (1-10 ng/band), nondestructive detection of gel-separated LPS,16,27 under fully reversible conditions for fast (10 min) and efficient (greater than 90%) passive elution of selected LPS bands, has been already established.28 This latter work,28 however, is limited only to the evaluation of the recovery of wellknown commercial LPS preparations in quantities of a few micrograms, which probably were originally depleted of impurities that may interfere with LPS biochemical analyses and might yet be present in samples from other sources. Therefore, the question still remained whether this isolation method would work well under routine experimental conditions to provide LPS fractions in enough quantities and with the necessary quality (e.g., purity and integrity) so as to allow satisfactory analysis of LPS by commonly used mass spectrometry methods. Here, we evidenced that while not compromising the unsurpassed separation that can be achieved by SDS- or DOC-PAGE and the high sensitive analysis attained by electrospray ionization (ESI)-mass spectrometry, the reverse staining of gel-separated LPS patterns followed by enhanced passive diffusion recovery of selected intact glycolipid bands can be successfully used to connect both analytical techniques. EXPERIMENTAL SECTION Reagents. Bacterial Strain, Growth Conditions, and LPS Extraction. The typeable H. influenzae strain RM.118 [Rd-] used in this study has been described earlier.29 In general, LPS was extracted from lyophilized bacteria by using phenol-chloroformlight petroleum, as described earlier.29 Procedure for LPS Micropurification. The method for micropurification of LPS consisted of four steps: preparative DOC-PAGE, reverse staining of gel-separated LPS, LPS mobilization, and LPS elution. Preparative DOC-PAGE. An aqueous suspension with 200 µg of crude LPS was prepared and electrophoresed in a glycine DOC-PAGE system as previously described,23 using a homemade single-slab gel apparatus. Gel dimensions were 17.5 × 35 × 0.2 (21) Steinberg, T. H.; Pretty On Top, K.; Berggren, K. N.; Kemper, C.; Jones, L.; Diwu, Z.; Haugland, R. P.; Patton, W. F. Proteomics 2001, 1, 841-855. (22) Lesse, A. J.; Campagnari, A. A.; Bittner, W. E.; Apicella, M. A. J. Immunol. Methods 1990, 126, 109-117. (23) Komuro, T.; Galanos, C. J. Chromatogr. 1988, 450, 381-387. (24) Noda, K.; Kubota, K.; Yamasaki, R. Anal. Biochem. 2000, 279, 18-22. (25) Inzana, T. J., Apicella, M. A. Electrophoresis 1999, 20, 462-465. (26) Davies, R. L.; Ali, Q.; Parton, R.; Coote, J. G.; Gibbs, A.; Freer, J. H. FEMS Microbiol. Lett. 1991, 69, 23-28. (27) Ferna´ndez-Patro´n, C.; Castellanos-Serra, L.; Hardy, E.; Guerra, M.; Este´vez, E.; Mehl, E.; Frank, R. W. Electrophoresis 1998, 19, 2398-2406. (28) Pupo, E.; Lopez, C. M.; Alonso, M.; Hardy, E. Electrophoresis 2000, 21, 526-530. (29) Risberg, A.; Masoud, H.; Martin, A.; Richards, J. C.; Moxon, E. R.; Schweda, E. K. H. Eur. J. Biochem. 1999, 261, 171-180.

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Figure 1. 18% DOC-PAGE separation of LPS-glycoforms from H. influenzae strain RM.118 [Rd-] and re-electrophoresis analysis of purified LPS bands. Gels were stained with zinc and imidazole,16 except in (D) which was stained with silver.1 A total of 200 µg of the crude LPS was applied to the preparative DOC-tris-glycine system and separated into two LPS bands; these are pointed to by arrows and designated as H and L bands. (A) Densitometric analysis of the gel is used to calculate the resolution of LPS bands. (B) and (C) are replicates of the preparative separation of the LPS by 18% DOC-PAGE. (D) and (E) The fast-migrating H and L bands in gels (B) and (C) were separately micropurified and re-electrophoresed in an analytical 18% DOC-PAGE. (D) Lanes (a) 2.7 µg of band L and (b) 1.3 µg of band H purified from gel in (B); (c) 5 µg of crude LPS were simultaneously run for comparison. (E) Lanes (a) 5 µg of crude LPS were run in parallel for comparison; (b) 3.6 µg of band H and (c) 3.1 µg of band L purified from gel in (C).

cm and 17.1 × 15.7 × 0.2 cm. The glycine DOC-PAGE system used a 3.5% stacking and a 18% separating gel. The sample was loaded onto a single, wide gel well and then run at 20 mA at room temperature until the dye had reached approximately 2 cm from the bottom of the gel. LPS Visualization. The polyacrylamide gel-separated LPS patterns were detected as transparent, colorless bands contrasting against a white gel background by staining with zinc-imidazole as described earlier.20 Negatively stained bands were clearly observable by placing the gel above any dark background. Clear gel images, such as those shown in Figure 1, could be easily stored by scanning the stained gel with a ScanJet 4c/T scanner (Hewlett-Packard Co., Palo Alto, CA) in a transillumination mode. To do this, the gel was placed on the scanner glass, and the scanning was done while keeping the scanner cover lifted and the lights off in the room. Stained gels could be stored, without fading, in water at 4 °C at least for a week. To estimate the resolution of LPS separation, a densitometric profile of the scanned gel image was obtained by using the Molecular Analyst software (Bio-Rad Laboratories, Hercules, CA). The resolution (Rs) between two peaks in the densitometric profile was calculated as follows: Rs ) 2Z/(wA + wB), where Z is the separation distance between peaks A and B, and wA and wB are the widths at the base of peaks A and B, respectively. Acceptable resolution is on the order of Rs ) 1, and baseline resolution between two peaks requires an Rs > 1.5. LPS Mobilization. Following detection, LPS bands of interest were separately excised and incubated (2 × 10 min) under agitation in 50 mL of 20 mM Tris-HCl (pH 8), 100 mM EDTA to chelate zinc ions, and washed (3 × 10 min) with 50 mL of water to thoroughly remove the chelating solution. LPS Elution. This procedure was carried out as described in ref 28 with few modifications. Briefly, gel slices were finely crushed to obtain gel microparticles with an average size of 32µm and collected in separate 50-mL tubes. To passively elute LPS 4920 Analytical Chemistry, Vol. 75, No. 18, September 15, 2003

molecules, 20 mL of 5% (v/v) triethylamine was added to the tubes, and the gel slurries were incubated under permanent vortexing for 10 min at room temperature. The tubes were centrifuged for 2 min at 1200 × g, and the overlaying solution was collected. This elution step was repeated once more. Samples containing the overlaying solution collected from these two LPS elution steps were pooled and repeatedly dried under vacuum at room temperature to remove triethylamine traces. Procedure for Purity and Recovery Analyses of Micropurified LPS. Analytical DOC-PAGE. LPS fractions were prepared for electrophoresis and separated in a glycine DOC-PAGE system as previously described,23 using a homemade (17.6 × 8.3 × 0.12 cm) single-slab gel apparatus. The glycine DOC-PAGE system used a 3.5% stacking and a 18% separating gel. Aliquots of 10-20 µL were loaded onto gel wells and run at 15 mA/slab gel until the dye had migrated 15 cm. Quantification of Recovery and Purity of LPS Fractions. Purified LPS fractions were subjected to analytical DOC-PAGE as described above followed by gel staining with silver.1 As a standard, a fixed amount of crude LPS was electrophoresed in a parallel lane. The quantity of each purified LPS fraction was estimated by comparison of the optical density of its corresponding silver-stained lane with that of the lane corresponding to the standard. LPS recovery was calculated as the ratio of the total quantity of each purified LPS fraction to the total quantity of crude LPS applied to the preparative gel. Purity of LPS fractions was estimated by densitometric analysis of analytical silver-stained gels. The densitometric analysis was performed with the aid of a ScanJet 4c/T scanner (Hewlett-Packard Co., Palo Alto, CA) and Molecular Analyst software (Bio-Rad Laboratories, Hercules, CA). Procedure for Electrospray Ionization (ESI)-Mass Spectrometry Analysis of LPS and Micropurified LPS Fractions. Preparation of Lipopolysaccharides. O-Deacylation of LPS was achieved as previously described using anhydrous hydrazine.29

Table 1. Recovery of the Mass of the High- and Low-Molecular-Weight LPS-Glycoforms from H. influenzae Strain RM.118 [Rd-]sPointed to by Arrows in Figure 1sby Preparative-Slab-PAGE Micropurificationa recovery (%) with respect to the total initial mass of

crude LPS higher-molecular-weight band of crude LPS lower-molecular-weight band of crude LPS purified higher-molecular-weight fraction purified lower-molecular-weight fraction total purified (higher- and lower-molecular-weight) fractions

mass (µg)

crude LPS

200 77.7 (6.46; 8.3) 122.3 (6.38; 5.2) 10 (0.5; 5) 50 (35.4; 70.8) 60 (42.5; 70.9)

5 (0.25; 5) 25 (17.7; 70.8) 30 (17.7; 59)

higher-molecularweight band of crude LPS

lower-molecularweight band of crude LPS

12.8 (1.07; 8.3) 40.9 (28.96; 70.8)

a This was determined as described in the Procedure for purity and recovery analyses of micropurified LPS under the Experimental Section. Values represent the average followed by the standard deviation and coefficient of variation in parentheses in a set of four independent experiments.

Prior mass spectrometry, LPS samples (62 µg LPS, 20 µg O-deacylated LPS (LPS-OH) of L and 10 µg LPS of H fractions, respectively) were transferred to an eppendorf tube and subjected to mild acid hydrolysis (1% HOAc, pH 3.1, 2 h, 100 °C). After lyophilization, the products were treated with sodium borohydride (10 mg, 2 h, 20 °C) in ammonia (1 M, 100 µL). The reaction was quenched with glacial acetic acid (100 µL) and evaporated. The boric acid was eliminated by evaporating with 10% acetic acid in methanol (200 µL) and thereafter methanol (2 × 200 µL). The products were treated with aqueous fluoric acid (48%, 48 h, 4 °C) and evaporated with a stream of nitrogen at 0 °C. The entire content of the tube was transferred to a glass vial and permethylated with methyl iodide (0.1 mL) in dimethyl sulfoxide (0.25 mL) in the presence of lithium methylsulfinylmethanide.30 The resulting methylated oligosaccharides were recovered on a SepPak C18 cartridge, concentrated to 150 µL, and subjected to ESI-MS/ MS analyses. Mass Spectrometry. Electrospray ionization mass spectrometry (ESI-MS) and tandem ESI-MS (ESI-MS/MS) experiments were performed on a Finnigan LCQ iontrap mass spectrometer (Finnigan-MAT, San Jose, CA). All experiments were done in the positive mode. LC-ESI-MS and LC-ESI-MSn experiments were carried out on a Waters 2690 HPLC system (Waters, Milford, USA) coupled to the mass spectrometer. A microbore C18-column (Phenomenex LUNA 5u C18 [2]) was used with an eluent gradient consisting of 0.01 M NaOAc and 1% HOAc in MeOH as eluent A and 0.01 M NaOAc and 1% HOAc in H2O as eluent B. A gradient program was used starting with 50% A rising to 100% A in 50 min and thereafter 100% A for 20 min. The flow rate was 150 µL/min. RESULTS AND DISCUSSION This work had as a main goal to ascertain that ESI-mass spectrometry analysis can be advantageously linked to LPS separation by slab-PAGE to directly determine the molecular masses of gel-separated LPS glycoforms and perform their sequence analyses. To answer this question, the previously characterized LPS from H. influenzae strain RM.118 [Rd-] was used as a model,29 and the following steps had to be carried out first: (i) to establish electrophoretic separation conditions for adequate resolution of H. influenzae LPS-glycoforms, (ii) to apply our previously described micropurification method,28 for the (30) Blakeney, A. B.; Stone, B. A. Carbohydr. Res. 1985, 140, 319-324.

recovery of the gel-separated LPS bands, as well as to determine the (iii) purity and (iv) integrity of purified LPS samples. Micropurification of LPS-Glycoforms. Polyacrylamide Gel Electrophoretic Separation. The electrophoretic separation of 200 µg of the LPS from H. influenzae strain RM.118 [Rd-] was carried out using a 18% DOC-PAGE at concentrations of DOC previously shown to efficiently dissociate LPS molecules.23 The resulting LPS banding pattern consisted reproducibly of two low-molecularweight bandsspointed to by arrows in Figure 1, parts B and C, and designated as high (H) or low (L) bands. The relative contribution to total LPS mass of the high and low bands was 38.8% (CV ) 8.2%, n ) 4) and 61.1% (CV ) 5.2%, n ) 4), respectively. Upon densitometry analysis (R ) 0.7, Figure 1A), it was revealed that these two LPS bands were not completly, yet acceptably resolved in the gel. However, an actually higher resolution of LPS bands may not be discarded since the high sensitivity of LPS detection with zinc and imidazole (in the range of nanogram/band) might lead to a saturation of the optical density in these detected high-quantity LPS bands. In fact, as described below, LPS fractions obtained by elution of these LPS bands displayed a high purity. Recovery of Passively Eluted LPS Glycoforms. The two fast migrating H and L bands were recovered from the preparative gel with a reproducibly high purity (Figure 1, parts D and E, of 97.4% (CV ) 0.1%, n ) 4) and 98% (CV ) 0.3%, n ) 4) for the high and low bands, respectively). In addition, the integrity of micropurified LPS fractions was preserved as evidenced by reelectrophoresis of the freshly micropurified bands (Figure 1, parts D and E). In Table 1 the mass recovery values of the micropurified LPS fractions are presented. Notice that in an average LPS micropurification experiment 10 µg (CV ) 5.0%, n ) 4) of the H band and 50 µg (CV ) 70.8%, n ) 4) of the L band were obtained. The lower quantities of micropurified H band as compared to the micropurified L band correlated with a lower contribution of the H band to the total mass in the crude LPS (Figure 1, parts B and C). This observation illustrates how the properties of a particular LPS mixture (e.g., number of glycoforms and relative contribution of component glycoforms to total mass of the LPS mixture) may significantly alter the final mass recovery of the micropurified LPS fractions. Analytical Chemistry, Vol. 75, No. 18, September 15, 2003

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Figure 2. Positive ion ESI-MS spectrum of dephosphorylated and permethylated LPS of H. influenzae strain RM.118 [Rd-] (A), LPS corresponding to L fraction (B), and LPS corresponding to H fraction (C). Ions corresponding to [M + Na]+ of the Hex1 to Hex8 glycoforms are indicated.

Slab-DOC-PAGE-based separation of the LPS fractions as described above has several advantages. These include its unsurpassed resolution combined with higher reproducibility, rapidity, lower cost, and simplicity. Electrospray Ionization (ESI)-Mass Spectrometry Analysis of Micropurified LPS Fractions. The major LPS structures in H. influenzae strain Rd have been established by us using NMR and ESI-MS on LPS-OH and major core oligosaccharides obtained after mild acid hydrolysis of LPS.29 In addition, minor components in the LPS containing sialylated31 or PEtn-GalNAc (unpublished results) capped lacto-N-neotetraose structures were recently identified and characterized (unpublished results). In those studies, ESI-MS spectra were obtained on LPS-OH in the negative mode. The amounts of isolated material from DOC-PAGE here were insufficient to give decent ESI-MS spectra (data not shown) when transformed to LPS-OH. We thus employed a strategy using (31) Cox, A. D.; Hood, D. W.; Martin, A.; Makepeace, K. M..; Deadman, M. E.; Li, J.; Brisson, J.-R.; Moxon, E. R.; Richards, J. C. Eur. J. Biochem. 2002, 269, 4009-4019.

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ESI-MS/MS on dephosphorylated and permethylated oligosaccharides derived from LPS.32,33 In general, permethylation increases the MS response by several orders of magnitude in all ionization modes. In addition, sequence information is readily obtained since methyl tagging allows the distinction between fragment ions generated by cleavage of a single glycosidic bond and inner fragments resulting from the rupture of two glycosidic linkages. The method here involved mild acid hydrolysis of LPS or LPS-OH, reduction and dephosphorylation of resulting oligosaccharide material in one reaction vial prior permethylation. We first applied this strategy to crude LPS from H. influenzae strain RM.118 (Rd-) for later comparison with the isolated materials from DOC-PAGE. Thus LPS was treated in an eppendorf tube as described above, transferred to a glass tube for the permethylation step, and (32) Reinhold V. N.; Reinhold: B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772-1784. (33) Månsson, M.; Hood, D. W.; Moxon, E. R.; Schweda, E. K. H. Eur. J. Biochem. 2003, 270, 610-624.

Table 2. Hex1-Hex8 Isomeric Glycoforms Observed in H. influenzae Strain RM.118 (Rd-) as Identified by Tandem LC-ESI-MS (ESI-MS/MS) after Permethylationa

a

Indicated are product ions of significant importance and the corresponding fragments.

subjected to ESI-MS in the positive mode. Figure 2A shows the resulting spectrum in which a major doubly charged ion corresponding to a sodiated permethylated Hex3 glycoform (composition Hex3Hep3AnKdo-ol) was observed at m/z 1671.9. Minor ions correspondingtocompositionsHex2Hep3AnKdo-ol,Hex4Hep3AnKdo-

ol, Hex4HexNAcHep3AnKdo-ol, Hex5HexNAcHep3AnKdo-ol, and Hex5HexNAc2Hep3AnKdo-ol were identified at m/z 1467.8, 1875.9, 2121.5, and 2325.7. MS2 experiments were performed on all molecular ions to get information on sequence. The obtained data are summarized in Table 2. For the Hex2 glycoform two isomeric Analytical Chemistry, Vol. 75, No. 18, September 15, 2003

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forms were identified of which one was in agreement with the earlier reported structure.29 It was defined by ions m/z 1001.5, 753.4, and 737.5 due to losses of terminal Hex-Hep, Hex-Hep-Hep, and Hex-Hep-AnKdo-ol units, respectively. The second isomeric form was defined by the ion at m/z 941.4 corresponding to the loss of a terminal HepAnKdo-ol unit. Similarly, the Hex3 glycoform was defined by ions m/z 1001.6, 753.5, and 941.5 due to losses of terminal Hex-Hex-Hep, Hex-Hex-Hep-Hep, and Hex-Hep-AnKdool. An ion at m/z 693.4 corresponded to the loss of a branched Hex-Hep(Hep)-AnKdo-ol unit. By analogy tandem MS experiments gave evidence for the Hex4 (two isomeric forms) and Hex5 (one isomeric form) glycoform structures. The Hex6 glycoform with the composition HexNAc‚Hex5‚Hep3‚AnKdo-ol was elongated by two hexoses from HepIII evident, inter alia, from the ion at m/z 1654.7 due to the loss of tHex-Hex-Hep. The Hex7 glycoform with the composition HexNAc2‚Hex5‚Hep3‚AnKdo-ol was elongated by two hexoses from HepIII evident, inter alia, from the ion m/z 1899.5 (loss of tHex-Hex-Hep) and no elongation from HepII due to the ion at m/z 1652.7 (loss of tHex-Hex-Hep-Hep). Accordingly, HepI was elongated by a HexNAc2Hex3 unit. Thus it was shown that the LPS from H. influenzae strain RM.118 [Rd-] not only contained the glycoforms identified earlier but also minor components that either were isomeric glycoforms or glycoforms of higher molecular weight not easily detected by conventional methods. When the H. influenzae LPS was subjected to micropurification assisted by DOC-PAGE two bands were recovered (see above) which respectively corresponded to the low- (0.075 mg) and high- (0.010 mg) molecular-weight fractions. We subjected the isolated bands (20 µg of L and 10 µg of H) to the microderivatization strategy followed by LC-ESI-MS, and the respective spectra of Figure 2, parts B and C, were obtained. The spectrum corresponding to the lower-molecular-weight band showed a major ion at m/z 1672.1 corresponding to the Hex3 glycoform. In addition, two minor ions at m/z 1468.1 and 1264.2 were observed corresponding to Hex2 and Hex1 glycoforms, respectively. MS/MS experiments evidenced one isomeric form for the Hex3 glycoform and two isomeric forms of the Hex1 glycoform (Table 2). In the spectrum corresponding to the higher molecular band (2C) ions corresponding to Hex2 to Hex5HexNAc2 glycoforms were observed at m/z 1468.3, 1671.9, 1875.6, 2121.6, 2325.4, and 2569.1. In addition, an ion corresponding to Hex6HexNAc2 was detected at m/z 2773.9. Although present, the ions of the lower glycoforms were minor abundant. The ions corresponding to the higher glycoforms were predominant with the ion at m/z 2325.4 (Hex5HexNAc) mostly pronounced. Further fragmentation of these ions revealed isomeric glycoforms identified above. For the Hex6HexNAc glycoform two isomeric forms were identified (Table 2). It was just evident that the two bands isolated after DOC-PAGE each consisted of several glycoforms. However, a clear separation between higher- and lower-molecularweight glycoforms had been achieved, and the amounts of recovered material were sufficient to allow detection and MS/ MS experiments for structural information. It should also be noted that mass values of the micropurified LPS bands (Figure 2, parts B and C) confirmed, within experimental error, those obtained

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Analytical Chemistry, Vol. 75, No. 18, September 15, 2003

by MS analysis of the crude LPS (Figure 2A). This further indicated that the integrity of micropurified LPS bands was preserved. In addition, analysis by ESI-MS of micropurified LPS samples in quantities similar to those of the crude LPS, a strategy called “trace enrichment”, led either to a similar intensity of the ions of the micropurified lower-molecular-weight LPS sample (Figure 2B) or a significantly higher intensity of the ions of the micropurified higher-molecular-weight LPS sample (Figure 2C), as compared to the same ion peaks of the crude LPS (Figure 2A), thus resulting in a higher sensitivity of ESI-MS analysis of less abundant higher-molecular-weight LPS-glycoforms. Moreover, micropurification of LPS fractions allowed detection of ions corresponding to the Hex1 (Figure 2B) and Hex6HexNAc2 (Figure 2C) glycoforms that were not detected by analysis of the crude LPS (Figure 2A). CONCLUSIONS We conclude that the present micropurification method is compatible with the analysis of LPS by ESI-MS. This was possible by the use of dephosphorylated and permethylated oligosaccharides derived from the micropurified LPS,32,33 which increased the sensitivity of ESI-MS analysis. To our knowledge, this is the first demonstration that slab-PAGE-based micropurification of LPS and LPS mass spectrometry analysis can be efficiently connected. Although the recovery percent of LPS mass of this micropurification method is lower (30%) than that of its analytical version (>90%),28 this methodology enables both a higher LPS mass load and LPS mass recovery, thus facilitating LPS analysis by commonly used mass spectrometry methods. Micropurified LPS glycoforms are in a highly purified state, essentially free of bacteria- or purification-associated pollutants (e.g., phospholipids, buffers, salts, detergents) or contaminated by other interfering LPS fractions. However, further improvements of this micropurification method may be envisioned to fulfill the requirements of a routinary method for efficiently providing intact and pure LPS fractions for ESI-mass spectrometry analysis. These may include lowering of the number of steps (e.g., by direct ESI-MS analysis of crushed and triethylamine-incubated LPS bands), optimizing further the recovery of purified-LPS mass or adapting the micropurification method for ESI-MS analysis of LPS isolates in quantities much lower than those used in this work. In fact, after slab gel electrophoresis as low as nanograms of LPS per band can be detected,16 and would be quantitatively recovered, as previously demonstrated for 10-µg amounts of reverse-stained LPS.28 The use of the here described methodology will be very helpful in the analysis by ESI-mass spectrometry of different LPS glycoforms from complex mixtures of not only H. influenzae but other mucosal pathogens. Since slab-PAGE-purified LPS have shown to be biologically active,14,20,28 mass-spectrometry-characterized LPS fractions may be concurrently used in biological assays for unambiguous determination of LPS structure-function relationships. Received for review April 9, 2003. Accepted July 11, 2003. AC034365O