Biochemical and Biotechnological Applications of Electrospray

Chapter 8

Determining Structures and Functions of Surface Glycolipids in Pathogenic Haemophilus Bacteria by Electrospray Ionization Mass Spectrometry

Downloaded by CORNELL UNIV on August 28, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch008

Bradford W. Gibson, Nancy J. Phillips, William Melaugh, and Jeffrey J. Engstrom Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, CA 94143-0446 An abundance of immunochemical and biological data implicates bacterial glycolipids, or lipooligosaccharides (LOS), in the diseases caused by pathogenic strains of Haemophilus and Neisseria species. These LOS consist of a lipid A attached to a variable and sometimes highly branched oligosaccharide region. There are various reasons why electrospray ionization mass spectrometry (ESI-MS) is an ideal tool for investigating the structure/function of bacterial LOS. First, LOS are biosynthesized as heterogeneous populations that cannot be readily separated, and mass spectrometry is well-suited for mixture analysis. For example, ESI-MS and ESI-tandem mass spectrometry have been used to determine the molecular weights and partial structures of LOS containing as many as twenty distinct components isolated from a single bacterial strain. Second, in the determination of the structure/function relationship of biological molecules, one would like to focus on LOS most relevant to the actual disease states. To conduct structural characterization studies under biologically relevant conditions requires working with very small amounts of sample, consistent with the sensitivity of mass spectrometry. In this chapter, we will present examples which demonstrate the application of ESI-MS in characterizing LOS from pathogenic strains of Haemophilus. Pathogenic bacteria from Neisseria and Haemophilus are responsible for a large number of human diseases, including meningitis, gonorrhea, pneumonia, otitis media and chancroid. Over the last ten years, an abundance of immunochemical, chemical and biological data has been reported that has begun to show a clear link between the outer-membrane glycolipids of these bacteria and specific pathogenic processes contributing to these human diseases. These glycolipids, or lipooligosaccharides (LOS), consist of a largely conserved lipid A region linked to a highly variable, branched and complex set of oligosaccharide moieties (see Figure 1). The lipid A is made up of a hexaacyl-substituted glucosamine disaccharide containing two phosphates that is attached to the oligosaccharide region through an acidic sugar, 2keto-3-deoxy-mannooctulosonic acid (KDO). The LOS from Haemophilus and Neisseria differfrommost enteric bacterial surface glycolipids, or lipopolysaccharides (LPS), by their much smaller size (M « 3500-6000), variable composition, antigenic diversity, high cytotoxicity, and lack of repeating terminal structures. r

0097-6156/95/0619-0166$12.00/0 © 1996 American Chemical Society

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

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Surface Glycolipids in Pathogenic Haemophilus Bacteria

The lack of repeating terminal saccharides in LOS is the most dramatic difference between LOS and LPS, although the remaining oligosaccharide regions of LOS represent an even more important structural difference when compared to the analogous core oligosaccharides of LPS. The LPS structures of the enteric bacteria such as Salmonella typhimurium and Escherichia coli (7,2), for example, contain relatively conserved core oligosaccharide regions that are extended by the addition of an O-antigen, a polysaccharide made up of repeating oligosaccharide units, neither of which bear much resemblance to mammalian sugar sequences. In contrast, LOS structures from H. influenzae, H. ducreyi and M gonorrhoeae have been shown to contain sugar sequences that resemble those present in human glycosphingolipids, such as lactose, N-acetyllactosamine, lacto-N-neotetraose, N-acetyl sialyllactosamine (NeuAc3Ga^l-»4GlcNAc) and the P antigen, Galal-^Gaipi-^Glc (for review, see (5)). In H. ducreyi, LOS itself has been shown to be highly cytotoxic in animal models, forming necrotic skin lesions that resemble the chancroid ulcers found in the human disease (5). Moreover, in a process termed 'phase variation' (5,6), LOS populations can change in structures and/or in relative proportions, making these nonenteric bacterial pathogens highly adaptive to the changing and hostile environment typical of that encountered in the human host. One of the first goals towards elucidating the roles that LOS play in hostpathogen interactions is to determine the precise LOS species expressed by these bacteria, preferably under the conditions of the disease process itself. Elucidating LOS structures expressed in vivo will require major advances in both the detection and sample preparation protocols over what is currently available. While there are a number of techniques used by chemists to characterize the structures of LOS and LPS, virtually all of these methods suffer serious shortcomings in regards to detennining the precise heterogeneity of LOS glycoforms. Most of these techniques, for example, require chemical treatment of LOS, such as mild acid hydrolysis to generate a water soluble oligosaccharide fraction and a chloroform/methanol soluble lipid A fraction. Indeed, the vast majority of chemical and structural studies of LOS and LPS has been on these oligosaccharide and lipid A fractions. Although mild acid hydrolysis of LOS and LPS is designed specifically to cleave at the labile glycosidic bond between the core KDO and lipid A moiety (see Figure 1), this procedure can also remove other acid labile moieties such as sialic acid or result in the β-elimination of specific phosphate groups on KDO (7). Furthermore, heterogeneity can be artificially generated in the lipid A portion as well, givingriseto monophosphoryl lipid A species and/or lipid A species lacking one or more 0-acyl substituted fatty acids moieties. Even treatment with aqueous HF under conditions designed to selectively remove phosphate groups can produce artifactual LOS species by cleaving glycosidic bonds. Given this situation, we have sought to develop mass spectrometric-based procedures that will provide an accurate as possible assessment of LOS ^heterogeneity without introducing artifacts or losing important structural information due to chemical degradation of specific functional groups. While we have used several mass spectrometric techniques to analyze LOS and their oligosaccharide and lipid A moieties, there are a number of reasons why electrospray ionization mass spectrometry (ESI-MS) is well-suited to investigating the structure/function of bacterial LOS. First, ESI-MS is an excellent technique for complex mixture analysis under conditions where one sees a somewhat restricted number of charge states per molecular species. Second, given the presence of several phosphate groups on the LOS, the fixed negative charges ensure excellent ionization in the negative ion mode and within a mass range where they can be readily observed as their major doubly or triply charged species. Third, the low background and large dynamic range afforded by ESI-MS allow for relatively minor species to be detected even in the presence of LOS species of considerably greater abundance. Fourth, in the determination of the structure/function relationship of biological molecules, one would like to focus on LOS species most relevant to the actual disease states. ESI-MS k


167



Figure 1. LOS structures found in (A) Haemophilus influenzae strain 2019 (7) and (B) Haemophilus ducreyi strain 35000 (19,20). Note the terminal sugars in these LOS constitute lactose or N-acetyl sialyllactosamine, structures common to many human glycoconjugates. The position of the phosphoethanolamine (PEA) group is not known but has been presumed to be linked to one of the core heptoses. However, more recent unpublished data suggests an additional PEA group is lost during mild acid treatment and may be linked to the phosphate of KDO. Abbreviations for sugars used in the figure and throughout the text are as follows: Gal is galactose, Glc is glucose; GlcN or GlcNAc is N-acetylglucosamine; Heptose is L-glycero-O-manno heptose, KDO is O-manno-2-keto3-deoxy octulosonic acid; NeuAc is N-acetylneuraminic acid (or silaic acid); KDO(P) is 4-phosphoKDO. Hep* refers to an unusual heptose, O-glycero-Omanno heptose.

(A) Haemophilus influenzae LOS


GIBSON ΈΤ AL.

Surface Glycolipids in Pathogenic Haemophilus Bacteria 169


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170

BIOLOGICAL AND BIOTECHNOLOGICAL APPLICATIONS OF ESI-MS

appears to have the necessary high degree of sensitivity to conduct structural characterization studies under biologically relevant conditions that may require working with very small amounts of sample, perhaps as little as a few femtomoles. Recent advances in ESI-MS sample introduction techniques such as the nanoliter spray methods (8) (see Chapter 2 of this volume) look particularly encouraging in this regard. In this chapter, we will discuss some of our most recent efforts to analyze LOS by ESI-MS, either as fully intact species or as their O-deacylated derivatives. For a discussion on the ESI approaches for the analysis of lipid A, one should see Chapter 9 of this volume and two recent papers (9JO). Our discussion, however, will emphasize methods for determining the structures of the variable oligosaccharide portions from pathogenic Haemophilus LOS and will be primarily limited to molecular mass determinations of LOS in complex mixtures (11). A few examples, however, will be presented that address strategies for limited structure determination of LOS by using ESI-tandem mass spectrometry or treatment with glycosidases in combination with ESI-MS. Methods. Isolation and Purification of LOS and O-deacylated LOS. LOS from H. ducreyi strains 35000 and 188-2 were isolated using a modified phenol/water extraction procedure of Westphal and Jahn (12). The LOS from H. influenzae strain A2 was prepared by the procedure of Darveau and Hancock (13). For mass spectrometric analysis, small amounts of LOS were O-deacylated according to a modified procedure of Helander et al. (14). Briefly, 0.5-1 mg of LOS was incubated with 200 of anhydrous hydrazine for 20 min at 37°C. The samples were then cooled to -20°C and chilled acetone was added dropwise to precipitate the O-deacylated LOS which was then centrifuged at 12,000 χ g for 20 min. The supernatant was removed and the pellet washed again with cold acetone and centrifuged. Precipitated O-deacylated LOS was then resuspended in 500 of water and lyophilized. Electrospray Ionization Mass Spectrometry (ESI-MS). For negative-ion ESI-MS analysis, either a VG-Fisons Platform quadrupole or a VG-Fisons Bio-Q triple quadrupole mass spectrometer with an electrospray ion source was used to mass analyze unmodified LOS and 0-4eacylated LOS. For the high resolution analysis of O-deacylated LOS from H. influenzae strain A2, a VG-Fisons AutoSpec magnetic sector instrument operating at a resolving power of 1000-3000 Am/m was used with an electrospray ion source. For these ESI-MS experiments, the O-deacylated LOS samples were first dissolved in water to make a 1 μg/μL solution. One μ ί of this solution was mixed with 4 μL of the running solvent and the 5 \\L was injected via a Rheodyne injector into a constant stream of H2O/CH3CN (3:1) containing 1% acetic acid running at 10 uiymin. For the analysis of unmodified H. ducreyi 35000 LOS, a similar procedure was followed except that the samples were first dissolved in H20/CH3CN/triethylamine (1/1/1, v/v) to make a 1 μg/μL solution, and the running solvent was H2O/CH3CN (1/1, v/v) containing 1% acetic acid. It was important to prepare fresh solutions of LOS prior to the run, since degradation occurred if the sample was allowed to remain in solution for prolonged periods of time. Mass calibration was carried out with an external horse heart myoglobin reference using the supplied VG-Fisons software. Electrospray tandem mass spectrometric studies of O-deacylated LOS were performed on a VG-Fisons BioQ triple quadrupole mass spectrometer. Conditions were similar to those mentioned above for ESI-MS of O-deacylated LOS. For the tandem experiments argon was used as the collision gas. The argon gas pressure in the collision quadrupole chamber was 10 torr and the collision energy was 17V. -2


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Results and Discussions. Over the last five years we have determined the structures of LOS from several pathogenic species of Neisseria and Haemophilus using liquid secondary ion mass spectrometry (LSIMS), tandem mass spectrometry, methylation analysis and NMR techniques (7,15-20). More recently, we have developed ESI-MS techniques as an integral part of our LOS structural studies (77). Other groups have reported structures and/or mass spectrometric methods for the analysis of the largely invariant lipid A regionsfromthese same organisms (14,21,22). From these data, we have constructed a structural model for LOS that begins to explain why these organisms are effective at surviving in the body and colonizing specific host tissues (see Figure 1) (77). For example, these bacteria mimic host carbohydrate structures in their LOS, and may provide the bacteria a means either to attach to and/or invade host tissues, or to evade the immune system. Recent reports have shown that sialylation of lactosaminecontaining LOSfromNeisseria confers serum resistance and inhibits opsonization and killing by human neutrophils (23-26). As mentioned earlier, the analysis of LOS by ESI-MS techniques has generally required the conversion of LOS to their O-deacylated forms by hydrazine treatment (see Figure 2). This simple one-step procedure removes four of the six fatty acyl moieties on the lipid A, producing a water soluble LOS derivative that now contains only two N-linked fatty acids (β-hydroxy myristic acid) and threefreephosphates (two on lipid A and one on KDO for Haemophilus species). Although further treatment with aqueous HF can produce LOS derivatives that are now amenable to MS techniques that prefer less anionic forms for optimum ionization efficiency such as LSIMS or matrix-assisted laser desorption ionization (MALDI) mass spectrometry, HF treatment produces some degree of glycosidic bond cleavage which can yield truncated LOS species that can confound the determination of the true LOS-glycoform population. Therefore, our results and discussion will be limited to the analysis of Odeacylated and intact LOS species by ESI-MS. A summary of chromatographic and mass spectrometric strategies used and/or are under investigation for analyzing intact LOS, O-deacylated LOS, and O-deacylated and dephosphorylated LOS, is summarized in Figure 2. Determination of sialic acid in the LOS of H. ducreyi strain 35000. One of the first ESI-MS spectra taken of O-deacylated LOS was from H. ducreyi strain 35000 (77). Before this sample had been analyzed, we had previously determined the major oligosaccharide structure present in this LOS mixture after mild acid treatment using LSIMS and high-energy CID experiments (79), and later NMR (20): Galp 1 -»4GlcN Ac β 1 ->3Ga^ 1 -»4Hepa 1 ->6G\c$ 1 ->4Hepa 1 ->anhydroKDO 3 Τ Hepal-»2Hepal However, we were also aware that SDS-PAGE analysis of the total LOS pool had shown the presence of at least 3-4 additional glycoforms with both higher and lower molecular weights (27). The negative-ion ESI-MS spectrum of this O-deacylated LOS mixture shown in Figure 3, therefore, provided the first accurate molecular weight data that allowed us to properly assess the chemical nature of these less abundant glycoforms (11,20). In fact, it was relatively straightforward to make preliminary composition assignments of these additional LOS species based on simple extension or truncation of the oligosaccharide region of the major LOS-B form shown above. For example, a lower mass species (LOS-A) was present corresponding to the loss of a single hexose (Hex) residue, and three of the four higher mass species could be



172


NH NH 2

C

2

LOS

)

aqr.HF

O-deacylated LOS

Detergent soluble

Water soluble Low MeOH/CH CN

EDTA-H 0 2

3

Dephosphorylated, O-deacylated LOS MeOH and H 0 soluble 2

1 1 T E A / C H C N / H 0 (1/1/1) 3

ESI-MS MALDI-TOF?

SDS-PAGE electroelution

2

ESI-MS LSIMS MALDI-TOF

Capillary electrophoresis (off-line or on-line)

LSIMS MALDI-TOF

Reverse-Phase HPLC or High pH Anion Exchange Chromatography (HPEAC)

Figure 2. Analytical scheme for the preparation of LOS derivatives. Dashed arrows identify chromatographic interfaces to ESI that are currently under development in our group for LOS analysis. SDS-PAGE refers to sodium dodecylsulfate polyacrylamide gel electrophoresis.


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Surface Glycolipids in Pathogenic Haemophilus Bacteria 173

GIBSON ET AL.


(ΜΒ-3Η)3· B-3

LOS

Mt

A

2548.8 2711.4 2834.7 2913.9 3003.0 3127.2 3077.7

B= C D

E-3 1000.0

Ε F

(G)

D-3 970.3

Proposed Composition Β-Hex 3 Hex, HexNAc, 4 Hep, KDO(P), Lipid A' Β + PEA Β + HexNAc Β + NeuAc Β + NeuAc, PEA Β + Hex, HexNAc

(MB-2H)2B-2 1354.7

P3 1041.4

D" 1457.3 2

A"

800

850

900

950

2

C*

2

J £T-2

1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500

1550

Β

800

850

900

950

1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500

1550

Figure 3. Negative-ion ESI-MS spectra before (A) and after (B) treatment with neuraminidase to remove sialic acid (NeuAc) moieties from specific Odeacylated LOS glycoforms isolated from H. ducreyi strain 35000. Adapted fromref. (20).


174


(M -3H)-3 = D-3 866.1 D


LOS A Β C D Ε F G H I J Κ

Proposed Composition 2 Hex, 3 Hep, PEA, KDO(P) Lipid A' 3 Hex, 3 Hep, PEA, KDO(P), Lipid A' 3 Hex, 3 Hep, 2 PEA, KDO(P), Lipid A' 4 Hex, 3 Hep, PEA, KDO(P), Lipid A' 4 Hex, 3 Hep, 2 PEA, KDO(P), Lipid A' 5 Hex, 3 Hep, PEA, KDO(P), Lipid A* 6 Hex, 3 Hep, PEA, KDO(P), Lipid A' 7 Hex, 3 Hep, PEA, KDO(P), Lipid A* 8 Hex, 3 Hep, PEA, KDO(P), Lipid A' NeuAc, HexNAc, 5Hex, 3 Hep, PEA, KDO(P), Lipid A' NeuAc, HexNAc, 6Hex, 3 Hep, PEA, KDO(P), Lipid A'

-Me 2277.8 2438.4 2S61.1 2600.8 2723.1 2762.4 2925.9 3086.4 3249.0 3256.2 3416.4

t

B-3

811.6

F-3

919.8

I-3

1082

A-3

758.3

C-3

852.7

700

800

m/z

Figure 4. Negative ion ESI-MS analysis of LOS from H. influenzae strain A2 from a VG-Fisons BioQ quadrupole mass spectrometer at a resolution of «500 (Μ/ΔΜ). Note the lack of separation of the components LOS-I and LOS-J at «1082 and 1084.4, respectively (adapted after ref. (18)).


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Surface Glycolipids in Pathogenic HaemopMus lfcictena

175


assigned as extensions of the major LOS-B form by the addition of PEA (LOS-C), Nacetylhexosamine (HexNAc) (LOS-D), or Hex+HexNAc (LOS-F). However, the second most abundant species (LOS-E) had an unexpected mass 291 Da larger, which was recognized as the mass of sialic acid (N-acetylneuraminic acid, or NeuAc). Even though this species constituted a major percentage of the LOS species based on relative ion abundances, it went undetected in the previous oligosaccharide experiments presumably due to degradation during the mild acid treatment step. To confirm this tentative assignment, we simply treated this LOS mixture with neuraminidase to enzymatically remove the terminal α-linked sialic acid and re-ran the sample by ESI-MS. As shown in Figure 3b, the peaks assigned as LOS-E and LOS-F at m/z 1000 and 1041.4, respectively, which had been tentatively identified as sialic acid-containing LOS components disappeared, thus supporting the presence of a terminal sialic acid in these two LOS species. Analysis of LOS glycoforms from H. influenzae strain A2. Based on our initial results with the H. ducreyi 35000 LOS, a second study was carried out to evaluate a much more complex mixture of LOS species from a type b strain of H. influenzae. As with the previous example, the LOS from H. influenzae A2 had been shown to be highly heterogeneous by SDS-PAGE analysis («8 or more bands) (28), and contained perhaps twice as many distinct LOS species as seen in the H. ducreyi strain 35000. The negative ion ESI-MS spectrum was consistent with this initial assessment by SDS-PAGE, and ten distinct molecular species could be identified either as their doubly and/or triply charged ions, (M-2H) - and (M-3H) ' (see Figure 4). Based in part on LSIMS analysis of individual oligosaccharide masses determined from the hydrolyzed sample (18), compositional assignments were made based on the observed molecular weights, and a general structural formula could be constructed as follows, where the number of Hex moieties (x+y) on the two branches varied from 2-8: 2

3

(Hex) -»Hep-^KDO(P)->0-deacyl-Lipid A Τ (Hex) -*Hep--PEA Τ Hep x

y

In addition, a second set of LOS species were conjectured to exist based on the observed masses. These LOS species start out by assembling ifive hexose residues (x+y = 5), followed by the addition of HexNAc and sialic acid, presumably as a sialylated N-acetyl lactosamine structure. Although these tentative assignments could be made relatively easily, uncertainties and/or discrepancies in the observed masses compared to the expected mass values of these LOS were as large as ±1-2 Da, making these interpretations somewhat in doubt. Indeed, had it not been for the oligosaccharide data obtained previously from LSIMS analysis, it would have been impossible to make some of these interpretations. For example, for the less abundant LOS species, the precise charge state(s) could not be determined directly from the data due to the lack of a confirming doubly charged ion. This problem was particularly noted in the m/z 1100-1200 region where the triply charged peaks from the higher mass LOS (LOS-I, -J, and -K) begin to overlap with the doubly charged peaks from the lower mass components (LOS-A and -B), which was especially problematic for the (M-3H) " ion for LOS-K and the (M-2H) " ion for LOS-A. Furthermore, there was one ion series as noted above that seemed to indicate the presence of a divergent biosynthetic pathway leading to the synthesis of two N-acetyl sialyllactosamine terminating structures, LOS-J and -K. But in these cases, there was no evidence for the unsialylated N-acetyl lactosamine LOS species. This suggests that unlike that observed in H. ducreyi LOS, once N-acetyl lactosamine was formed as the presumed sialic acid acceptor, it was completely modified to the sialylated species, 3

2


176



865.9 (M -3H)3D

F-3 E-3

G-3 974.1

H-3 |-3 j-3

B-2 1218.0

D-2 1309.2 1375.5

900

950

ldoo

idso

1100 1150

idso

iio'o

1350 1400

Figure 5. Negative-ion ESI-MS analysis of H. influenzae A2 LOS from a magnetic sector VG/Fisons Autospec mass spectrometer at a resolution of 1000 and 3000 Μ/ΔΜ (inset). Note the separation of the individual isotopes in LOS-C at the higher mass resolving power.



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NeuAc->Ga^l-»4GlcNAc. Clearly, limitations of this relatively low resolving power quadrupole analyzer (R = «500 Μ/ΔΜ) were compromising our abilities to make accurate molecular weight and composition assignments. In an attempt to get around the limitations of mass accuracy and resolution, we investigated the utility of performing these experiments on a sector instrument with higher resolving power. And as we will see, improvements obtained on the AutoSpec magnetic sector instrument were impressive and offered several advantages (see Figure 5). First, the increased mass resolving power of the ESI-magnetic sector instrument provided much better separation of closely related LOS components. For example, the ion at m/z 866 can now be clearly assigned as a triply charged ion (see Figure 5, inset). In addition, the peaks at m/z 1082.2 and 1084.7 which were not fully separated in the previous quadrupole ESI-MS experiment (Figure 4) (18) are fully resolved at both the 1000 and 3000 Μ/ΔΜ resolution settings. Moreover, the higher resolving power reveals the isotope pattern in these two molecular ions and allows one to assign charge states in a completely unambiguous manner. This is especially important for LOS analyses since we generally see only one (and sometimes two) charge state per LOS component under negative-ion ESI conditions (primarily ζ = -3, corresponding to the number of phosphates) (77). The lack of a confirming ion series, therefore, can make it difficult to precisely determine the molecular weights as well as make accurate component identifications based on observations of single peaks in a complex spectrum. Therefore, better resolution MS data from the VG-Fisons AutoSpec instrument provided a higher degree of mass precision (and in this case, mass accuracy) in the assignments of the LOS components. For example, LOS-I and -J can be completely resolved and their masses determined to within ±0.1-0.2 Da of their C monoisotopic M (see Figure 6, Table I). 1 2

r

Table I. Masses of Haemophilus influenzae A2 O-deacylated LOS at «3000 M / A M Resolution Under Negative-ion ESI-MS Conditions Observed Calculated ΔΜ Proposed LOS Compositions C mass C mass A= 2275.3 0.4 2Hex,3Hep,PEA,KDO(P),LA* 2275.78 B= 2437.5 2437.84 0.3 3Hex,3Hep,PEA,KDO(P),LA C= 2559.8 2599.89 0.1 3Hex,3Hep,2 PEA,KDO,LA D= 2599.8 2599.89 0.1 4Hex,3Hep,PEA,KDO(P),LA E= 2723.0 2722.90 0.1 4Hex,3Hep,2 PEA,KDO(P),LA F= 2762.0 2761.94

Biochemical and Biotechnological Applications of Electrospray

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