SARAH ELIZABETH STEFAN

Transcription

1 DIFFERENTIATION OF CARBOYDRATE ISOMERS BY TUNABLE INFRARED MULTIPLE POTON DISSOCIATION AND FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY By SARA ELIZABET STEFAN A DISSERTATION PRESENTED TO TE GRADUATE SCOOL OF TE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF TE REQUIREMENTS FOR TE DEGREE OF DOCTOR OF PILOSOPY UNIVERSITY OF FLORIDA

2 2009 Sarah Elizabeth Stefan 2

3 To my Mom and Dad 3

4 ACKNOWLEDGMENTS Many people have supported and helped me throughout my graduate career. First, I would like to thank my parents. Their support and unconditional love have made my studies possible. I want to thank them for their understanding and encouragement when tough times occurred; I appreciate all their help and love more than they will ever know. I also thank my family for their support and for always making life interesting. I am grateful for my friends, old and new, who gave a helping hand and an ear for listening when I needed them. All the laughs and conversations over these past four years have lifted my spirits and helped me to keep going. I want to acknowledge my lab mates, past and present, for their help, knowledge and conversations have been instrumental in my work. I would also like to thank all my professors at Wheaton College, specifically Drs. Elita Pastra-Landis and Laura Muller, whose support and investment in me opened my eyes and mind to the potential of graduate school. Their enthusiasm and support have made all the difference. I have the deepest gratitude to all the people with whom I collaborated; they have made my project possible. First, I wish to thank my advisor, Dr. John Eyler, for his guidance, patience and support during my graduate career. I want to thank Dr. Brad Bendiak for all the samples, advice and support that he has provided throughout this project. is guidance and suggestions were well needed and helped tremendously. I would also like to thank Dr. David Powell for use of his instrument for the negative disaccharide work. Next, I want to thank my other committee members, Drs. Nicolo Omenetto, Nicolas Polfer and Carrie askell-luevano, whose questions and conversations have helped me along the way. Finally, I would like to thank Drs. Jos Oomens and Jeffrey Steill for their help and effort with the work performed at the Free Electron Laser for Infrared experiments (FELIX) facility. Without all of these people, this dissertation would not be possible. 4

5 Finally I need to thank the one person who has had to listen to me late at night and early in the morning, whose patience and loving shoulder made it easier to continue when I wanted to give up, Mr. Brad ouse. is immense computer knowledge and lack of chemistry knowledge helped me survive the past four years. 5

6 TABLE OF CONTENTS ACKNOWLEDGMENTS...4 LIST OF TABLES...9 LIST OF FIGURES...10 ABSTRACT...13 CAPTER 1 INTRODUCTION...15 page Carbohydrates...15 Monosaccharides...15 Disaccharides...18 Oligo- and Polysaccharides...19 Differentiation of Mono- and Disaccharides...21 Separation of Oligosaccharides...22 Analysis Methods...24 Mass Spectrometry: Ionization Techniques...26 Fragmentation Methods...27 Charged Ions...29 Objective of This Research...31 Overview FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY...39 istory...39 Apparatus...40 Magnet...40 Vacuum System...40 Analyzer Cell...41 Data System...41 Theory...42 Cyclotron Motion...42 Trapping Motion...43 Magnetron Motion...44 Basic FTICR-MS Operation and Data Acquisition...46 Mass Resolution...49 Tandem Mass Spectrometry...51 Dissociation Techniques...51 Conclusions

7 3 INFRARED MULTIPLE POTON DISSOCIATION...58 Introduction...58 Mechanism of Infrared Multiple Photon Dissociation...59 Lasers Used for IRMPD DIFFERENTIATION OF MONOSACCARIDES IN TE POSITIVE ION MODE BY IRMPD WIT A TUNABLE CO 2 LASER...66 Introduction...66 Procedure...67 Reproducibility...68 Results and Discussion...69 Methyl-glucopyranosides...69 Unknown Study of Methyl-glucopyranosides...71 Methyl-galactopyranosides...71 Unknown Study of both Methyl-gluco- and galactopyranosides...72 Conclusions DIFFERENTIATION OF DISACCARIDES IN TE POSITIVE ION MODE WIT A TUNABLE CO 2 LASER...83 Introduction...83 Procedure...84 Fragmentation Study...84 Anomeric Configuration Study...85 Results and Discussion...85 Differentiation of Disaccharides...85 Determination of the Anomeric Configurations...86 Differentiation of Unknowns...87 Conclusions IRMPD STUDIES OF NEGATIVELY CARGED DISACCARIDES WIT A TUNABLE CO 2 LASER...93 Introduction...93 Procedure...94 Deprotonated Disaccharides...94 Chlorinated Disaccharides...95 Reproducibility: Deprotonated Disaccharides...96 Reproducibility: Chlorinated Disaccharides...96 Results and Discussion...97 Deprotonated Disaccharides...97 Chlorinated Disaccharides...98 Identification of Fragment Ions Conclusions

8 7 DIFFERENTIATION OF DISACCARIDES IN TE NEGATIVE ION MODE WIT FREE ELECTRON LASER INFRARED MULTIPLE POTON DISSOCIATION Introduction Procedure Results and Discussion Disaccharides Monosaccharide Anion Produced from Disaccharides Conclusions CONCLUSIONS AND FUTURE WORK LIST OF REFERENCES BIOGRAPICAL SKETC

9 LIST OF TABLES Table page 5-1 Table of ratios used to determine the laser power used for fragmentation Major fragment ions observed for the chlorinated disaccharides when the precursor ion (m/z 377) was almost depleted by infrared mulitple photon dissociation (IRMPD) at μm Comparison of the fragments produced by collision induced dissociation (CID) and IRMPD for the chlorinated disaccharides

10 LIST OF FIGURES Figure page 1-1 Fischer projection for D- and L-glucose Example of the numbering system for the carbons of monosaccharides Fischer projections for the D-hexoses of the aldose family Anomers of D-glucose Inter-conversion of the ring structures for the 6-membered ring, pyranose, and the 5-membered ring, furanose, of D-glucose Examples of disaccharides composed of two glucose (Glc) monosaccharides Structures of two common oligosaccharide derivatives Typical steps for analysis of glycans Fragmentation nomenclature for oligosaccharides Possible fragmentation pathways for fragmentation by infrared multiple photon dissociation (IRMPD) Ion cyclotron motion Schematic diagram of the components of a Bruker 4.7 T FTICR (Fourier transform ion cyclotron) mass spectrometer Figures of merit for FTICR-MS as a function of magnetic field strength Two of the typical analyzer cells used for in FTICR mass spectrometers General schematic of a typical experimental sequence Various domains and spectra obtained from an FTICR-MS experiment Effect of number of data points acquired and Fourier transform on mass resolution Energy potential well Depiction of the IRMPD mechanism in polyatomic molecules Schematic of an undulator used for free elctrom lasers (FELs) Layout schematic of Free Electrom Laser for Infrared experiments (FELIX)

11 4-1 Structures of the O-methylated monosaccharides discussed in this chapter Experimental set up of the 4.7 T FTICR mass spectrometer Wavelength-dependent fragmentation patterns for the lithiated O-methyl-glucopyranosides for wavelength from 9.2 to 10.8 μm Infrared mulitple photon dissociation depletion spectra of the precursor ions (m/z 201) for both α- and β-o-methyl-glucopyranoside lithium cation complexes Comparison of the fragmentation of β-methyl-glucopyranoside at wavelengths and μm Relative percent abundance of fragment ions for both lithiated α- and β-o-methyl-glucopyranosides over the wavelength range from to μm Spectra of unknowns in single blind study of methyl-glucopyranosides at wavelength μm Fragmentation patterns over the wavelengths from 9.2 to 10.6 μm Ratio of m/z 169 to m/z 151 for α- and β-o-methyl-galactopyranoside Decision flowchart used to identify the different monosaccharide anomers Spectra of unknowns identified as galactopyranosides in single blind study obtained at wavelength μm Wavelength-dependent fragmentation for the various linked lithiated disaccharides Flow-chart depicting how linkage of the disaccharides was determined Flow-chart showing ratios of peak heights and values used to determine anomeric configurations Bar graphs comparing ratios from knowns and unknown lithiated glucose-containing disaccharides at the wavelengths 9.342, and μm Schematic drawing of the laser/mass spectrometer set-up used for the analysis of deprotonated disaccharides Relative percent abundance of the precursor ion (m/z 341) of isomaltose at selected wavelengths Wavelength-dependent fragmentation patterns for the various deprotonated disaccharides Ratio of m/z 161/179 for 1-3 and 1-6 linked disaccharides, showing that this ratio is not optimal for distinguishing the different anomers

12 6-5 Comparison of the fragmentation patterns of deprotonated isomaltose on two separate days Fragmentation spectra for the nearly depleted precursor ion (m/z 377) for the chlorinated disaccharides at μm Infrared multiple photon dissociation spectra for chlorinated isomaltose obtained at three wavelengths on two different days Average fragmentation spectra for the disaccharides at 9.342, and μm Decision flow chart used to identify disaccharide samples with unknown identities in a single-blind study Comparison of various ratios used to determine the anomeric configurations of the chlorinated disaccharides Identification of some of the fragment ions for the various linked disaccharides Schematic of the FTICR set-up at FELIX Infrared multiple photon dissociation fragmentation patterns over the wavelength range of 5.5 to 11 μm for the deprotonated 18 O-labeled disaccharides Fragmentation pattern of chlorinated unlabeled sophorose Comparison of the IRMPD spectra for O 18 -labeled sophorose and O 16 -chlorinated sophorose Comparison of the IRMPD spectra of the monosaccharide anions (m/z 179) produced by deprotonation of glucose and by fragmentation of a disaccharide by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) and CO 2 laser irradiation Schematic of the possible mechanism leading to the opening of the monosaccharide anion ring Infrared multiple photon dissociation spectra of various deprotonated monosaccharides Comparison of the IRMPD spectra for anomers of O-methyl-glucopyranoside to the spectrum of deprotonated glucose Comparison of the fragmentation patterns of the deprotonated monosaccharides over the wavelength range of 5.5 to 11 μm

13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DIFFERENTIATION OF CARBOYDRATE ISOMERS BY TUNABLE INFRARED MULTIPLE POTON DISSOCIATION AND FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY Chair: John R. Eyler Major: Chemistry By Sarah Elizabeth Stefan May 2009 Carbohydrates and their derivatives play a crucial role in many biological processes including fertilization, cell growth, inflammation and post-translational protein modification. The function of carbohydrates in these systems is closely related to their structure, including monosaccharide sequence, glycosidic linkage and stereochemistry. Unfortunately, the number of anomeric configurations and possible linkages between monosaccharide units makes analysis of carbohydrate structures complex. In order to shed light on these larger oligosaccharides, the fragmentation patterns and infrared multiple photon dissociation (IRMPD) spectra of various mono- and disaccharides were obtained and compared. For this work, various tunable infrared sources including a line-tunable continuous-wave carbon dioxide laser and a free electron laser (FEL) were used in conjunction with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The first three projects used a line-tunable carbon dioxide laser to fragment various mono- and disaccharides in both the positive and negative ion modes. In the first project, anomers of lithium-cation attached O-methyl-gluco- and galactopyranosides were fragmented. The identity and anomeric configuration of each monosaccharide was accurately determined by comparing 13

14 fragmentation patterns and ratios of certain fragments. A second project explored the fragmentation pattern of lithiated glucose-containing disaccharides having various linkages (1-2, 1-3, 1-4 and 1-6) and anomeric configurations (alpha and beta). Both the linkage and anomeric configuration of the various disaccharides were successfully identified based on their fragmentation patterns at several wavelengths. Next, irradiation of deprotonated and chlorinated glucose-containing disaccharides produced fragmentation patterns in which cleavage of the glycosidic bond resulted in major abundances of m/z 161 and 179 fragment ions. Along with differentiating the anomeric configuration for the chlorinated disaccharides, comparison of the abundances for major fragment ions also resulted in the positive identification of the linkages for both sets of disaccharides. Lastly, several deprotonated (negatively charged) mono- and disaccharides were fragmented with a FEL. The IRMPD spectra of the monosaccharide anions (m/z 179) from both the deprotonated monosaccharides and those isolated by fragmentation of various disaccharides were taken. A C-O stretching band characteristic of aldehydes was present in all spectra at ~1720 wavenumbers and gave spectroscopic evidence of the monosaccharide ring opening and therefore loss of anomericity. 14

15 CAPTER 1 INTRODUCTION Carbohydrates and their derivatives are biologically important. They participate in cellcell interactions and also act as target structures for microorganisms, toxins and antibodies. 1-3 Carbohydrates also interact with proteins and play a critical role in fertilization, cell growth, inflammation and post-translational protein modifications. 1,3-5 The simplest unit within these larger carbohydrates is that of the monosaccharide. When two monosaccharides are joined together, the result is a disaccharide. The disaccharide is the smallest saccharide unit which contains the glycosidic bond. Depending on the anomeric configurations of the monosaccharides that react, a disaccharide can either be α- or β-linked. The role of carbohydrates depends not only on the subunits of sugars which compose them, but also how these units are linked together. 6 Therefore, characterization of the both the anomeric configuration and the linkage of the different types of mono- and disaccharides is important. Carbohydrates Carbohydrates can be categorized based on their degree of polymerization. The smallest group is that of monosaccharides and their derivatives, all of which are not polymerized. The next category includes oligosaccharides, that have 2 to 10 degrees of polymerization. The last category is that of polysaccharides, that have greater than 10 degrees of polymerization. This chapter will discuss all the possible types of carbohydrates as well as give an overview of the methods used for carbohydrate analysis. Monosaccharides Monosaccharides are the smallest units that compose larger oligosaccharides. There are several types of monosaccharides and they all have the general formula of (C 2 O) n. Typically the more biologically common isomer of monosaccharides in nature is the D-isomer, but 15

16 L-isomers are also found. The monosaccharide isomer can be determined by drawing the Fischer projection. In the Fischer projection when the hydroxyl group on the highest numbered stereocenter is on the right, it is the D-isomer and when the hydroxyl group is on the left it is the L-isomer, Figure Since D-isomers of sugars are found much more frequently in nature, this dissertation will deal only with D-isomers. The carbons in monosaccharides are numbered sequentially starting with the end of the chain nearest to the carbonyl group, as seen in Figure 1-2. Carbon number 1, also known as the anomeric carbon, is where two monosaccharides can be joined together, through a glycosidic linkage or bond, to form larger oligosaccharides. The smallest possible monosaccharide has a backbone composed of only 3 carbon atoms, but 4, 5 and 6 carbons are other possible backbones. The names of these monosaccharides are trioses, tetroses, pentoses, hexoses, and heptoses, respectively. Monosaccharides that contain a keto group are called ketose whereas monosaccharides containing an aldehyde are called aldoses. Typically the names of the family and number of carbons are combined into one systematic name. For example, a monosaccharide containing both a 4 carbon backbone and an aldehyde group would be named an aldotetrose (aldo for the aldehyde group and tetrose for the 4 carbon backbone). For the aldose family, each of the eight D-aldohexoses differs in stereochemistry at carbon 2, 3 or 4 and has its own unique, common name, such as D-glucose, D-galactose, etc., as shown in Figure 1-3. When two monosaccharides only differ at one carbon position, they are epimers. Since they only differ in the position of the hydroxyl group on carbon number 4, D-glucose and D-galactose are an example of epimers from the aldose family. Monosaccharides can be found in either the open chain or ring form, but typically the ring form is more common. In solution, monosaccharides with a 5 or 6 carbon backbone can undergo 16

17 nucleophilic attack of the carbonyl carbon by one of the hydroxyl groups along the chain, resulting in a ring. Six-membered rings are called pyranoses and 5-membered rings are called furanoses. 8,9 At least four carbons and one oxygen are needed to form a furanose. Therefore, aldotetroses and higher aldoses and 2-pentuloses and higher ketoses can be found in the furanose ring. While monosaccharide rings can be either 5- or 6-membered, pyranosides are the most common form. When cyclic monosaccharides only differ by the position of the hydroxyl group on the anomeric carbon, they are anomers. If the hydroxyl group is axial relative to the plane of the ring then it is said to be in the α-position and if it is equatorial then it is in the β-position, Figure 1-4. The cyclic monosaccharides can interconvert between α- and β-anomers through a process known as mutarotation, Figure 1-5. During mutarotation, the ring opens into the chain form. Once in the chain form, a nucleophilic attack results in the formation of the β-anomer. Therefore, in solution there is an equilibrium mixture of all possible isomers including the furanose, pyranose, α-, β- and open chain forms of the monosaccharides. This equilibrium mixture is different for each monosaccharide, but for D-glucose it is approximately one-third α-anomer, two-thirds β-anomer and less than 1% of both the open and five-membered ring forms. 7 On the other hand, D-mannose has approximately 69% α-anomer and 31% β-anomer in solution, thus showing that the equilibrium doesn t always contain more of the β-anomer than the α-anomer. The two cyclic forms of D-glucose are known as hemi-acetals, which are formed by the reaction of the hydroxyl group on carbon number 5 and the aldehyde group. Typically any monosaccharide that contains a hemiacetal group is a reducing sugar and can react further. A reducing sugar is one that reacts with Tollens (Ag(N 3 ) 2 O) or Benedict s reagents (solution of 17

18 copper (II) sulfate, sodium carbonate, sodium citrate dihydrate and 2,5-difluorotoluene) to reduce either Ag +2 or Cu +2. If a sugar contains an acetal group then it cannot react with the Tollens or Benedict s reagents and it is called a non-reducing sugar. While hexoses are the most abundant sugars, there are a number of monosaccharide sugar derivatives that are naturally abundant and important. Some of these derivatives are N-acetylneuraminic acid (sialic acid), α-d-acetylgalactosamine and α-d-acetylglucosamine. These derivatives are found primarily in animals as the major components of glycoproteins and glycolipids. Disaccharides Disaccharides, the next largest saccharide are formed when a hydroxyl group of one monosaccharide reacts with the anomeric carbon of the other, Figure 1-6. The resulting bond is known as an O-glycosidic linkage. When two cyclic hexoses come together, a glycosidic linkage can occur at one of the five hydroxyl positions. This leads to numerous possible isomers with various linkages. Disaccharides are composed of a non-reducing monosaccharide that is fixed in the ring conformation and a reducing-monosaccharide that can interconvert between the α- and the β-configuration. Therefore, in solution, there will be a mixture of the α- and β-configurations of the reducing sugar of the disaccharide. While most sugars have a common, non-systematic name, there is a systematic nomenclature scheme for disaccharides. In it, the name of the first monosaccharide unit, its anomeric configuration and then the linkage followed by the second monosaccharide unit is given. For example, two glucose (Glc) units that are α- connected at the 1 and 6 carbon will be named glucose α1-6 glucose (Glcα1-6Glc), for which the common name is isomaltose. For larger oligosaccharides the nomenclature process is the same, but for each monosaccharide attachment the linkage and anomeric configuration followed by the monosaccharide is given. 18

19 For example a trisaccharide that has a glucose β-linked to carbon number 2 of a mannose (Man) monosaccharide which is α-linked to carbon number 4 of another glucose unit would be named Glcβ1-2Manα1-4Glc. When the anomeric carbons of both monosaccharide units are linked, the anomeric configuration of each saccharide is given. For example, sucrose is a disaccharide when the anomeric carbon of both the glucose and fructose (Fru) monosaccharide units are linked. For this, the systematic name would be Glcα1-2βFru. Since both anomeric carbons are linked in sucrose, it is a non-reducing sugar, unlike kojibiose (Glcα1-2Glc) and sophorose (Glcβ1-2Glc) that are examples of reducing sugars. Oligo- and Polysaccharides Oligosaccharides are the next largest saccharide chains that consist of 3 to 10 monosaccharide units linked together. Sugars that contain more than ten monosaccharide units are called polysaccharides. Oligo- and polysaccharides can be either homo- or heter-oligosaccharides. omo-oligosaccharides contain the same monosaccharide unit that repeats, whereas heter-oligosaccharides contain different monosaccharide units linked together. One homo-polysaccharide is starch, which can be found in foods such as potatoes. Starches characteristically have α1-4 linkage between two glucose units. 10 Other polysaccharides that do not have this linkage, also known as non-starch polysaccharides, can be found in foods such as bran, bananas and hazelnuts. Other common polysaccharides are cellulose and glycogen. Cellulose is a polysaccharide that contains several hundreds to thousands of β1-4 linked glucose units. It is the main component of the primary cell walls of plants and can be found in some algae. Glycogen is a glucose-polysaccharide that has a lot of branching and most commonly functions as short-term energy storage in animals. Common oligosaccharide derivatives are those of N-acetyl hexosamines, primarily N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc), Figure The 19

20 GlcNAc reducing end is linked to serine or threonine residues whereas the GalNAc reducing end is linked to asparagines. N-acetylglucosamine is a component of chitin and GalNAc is the terminal carbohydrate that forms the antigen of blood group A. N-acetylgalactosamine is also the first monosaccharide unit that connects to serine and threonine in glycosylation and is necessary for intercellular communication. Polysaccharides and oligosaccharides are also known as glycans. Glycosylation is a post-translational modification where oligo- and polysaccharides are linked to proteins and lipids, forming glycoconjugates. Glycosylation is one of the most common post-translational modifications for proteins and it is approximated that more than 50% of all proteins are glycosylated. 12 Linkages between a glycan and a protein form glycoproteins and those with lipids form glycolipids. The type of glycoprotein is determined by the linkage between the carbohydrates and the protein. Glycoproteins can be O- or N-linked. While N-linked are linked by a chitobiose (dimer of β1-4-linked glucosamine units) unit to an amide nitrogen of an asparagine residue, O-linked are linked to the oxygen of a side chain of an amino acid. 13,14 Typically the linkage is through a serine or threonine residue. N-glycosidic bonds are found in all nucleotides (the resulting sugar and nucleotide structures are called nucleosides, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA)). Unlike other oligosaccharides that are linked by oxygen bridges, RNA and DNA are polyesters that are linked by phosphate bridges. DNA is the largest known polymer with more than units found in human genes and the number of units found decreases as one goes down the evolutionary chain. 8 Another example of a polysaccharide with N-linkages is chitin. Chitin is a naturally occurring polysaccharide, composed of β1-4-linked N-acetyl-D-glucosamines, which is found in places like fungi and exoskeletons of arthropods 20

21 such as crustaceans. The three classes of glycoproteins are: N-glycosyl protein, O-glycosyl protein and N,O-glycosyl protein. Since multiple types of linkages (O- or N-linked) and anomeric configurations are possible, it is no surprise that many different isomers are possible. When attached to proteins (glycoproteins), oligosaccharides have been found to aid in a plethora of functions in the human body including cellular recognition, signaling, receptor binding and immune responses They also serve to influence folding, biological lifetime and recognition of binding partners for proteins. 17 Carbohydrates are also involved in the glycosyl phosphatidyl-inositol (GPI) anchor, by which proteins are attached to the plasma membrane and the oligosaccharides are linked to lipids which are attached to cell membranes. 18 In this process, a glycolipid can be connected to the C-terminus of a protein during post-translation modification. Since the biological role of oligosaccharides depends on the linkage, branching, configuration and saccharide units, being able to distinguish and differentiate the smaller mono- and disaccharides that compose larger oligosaccharides is very important. Due to the various linkages (carbons 1-6 of each monosaccharide unit), anomeric configurations (α- or β-) and monosaccharide units (any of the eight D-hexoses) there is a plethora of possible isomers, which makes analysis of carbohydrates a very difficult task. Differentiation of Mono- and Disaccharides Glycans must be isolated and prepared for analysis. The preparation method can include releasing the glycans, separating them and then finally analyzing them. Once separated common methods for analysis have included nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). Figure 1-8 shows a schematic of the different methods used for separating and analyzing saccharides. 21

22 Separation of Oligosaccharides Typically oligosaccharides can be released by several methods, either chemical or enzymatic. Enzymatic methods use a specific enzyme to pick out a particular substrate from mixtures. 14 Enzymes, for example glycosidase and galactosidase, are used to remove specific sugar residues sequentially from the non-reducing end. A chemical method for releasing glycans is an alkali-borohydride treatment which then can be followed by hydrolysis, with the resulting species then separated by high performance liquid chromatography (PLC) and/or gas chromatography (GC). 19,20 Once released, the oligosaccharides can then be separated. Methods for determining and separating mixtures of carbohydrates include thin layer chromatography (TLC), column chromatography methods (including gas chromatography, liquid chromatography, gas-liquid chromatography and high performance liquid chromatography) and capillary electrophoresis (CE). 13,14,21 Thin layer chromatography is a relatively cheap and inexpensive method for separating analytes. Microcrystalline cellulose and silica gel are two typical solid supports. Cellulose separation occurs by a liquid-liquid partition where the sugar of interest is distributed between the mobile phase and the cellulose-bound complex in water. The separation occurs based on the solubility of sugar in the eluent and how easily it can enter the solid support. Cellulose TLC has the same chromatographic characteristics as paper TLC, but allows for shorter elution time and increased sensitivity. Silica gel separation is similar to cellulose, but requires an additional adsorption component, typically an inorganic salt (phosphate, bisulfate). Numerous solvents are used to separate the various monosaccharides. igh performance liquid chromatography, gas-chromatography (GC) and gas-liquid chromatography (GLC) can also be used to separate components of mixtures. All of 22

23 these methods require somewhat expensive equipment. igh performance liquid chromatography is typically preferred for monosaccharide mixtures, oligosaccharide analysis and purification. Whereas GLC is limited to monosaccharide mixtures only, PLC requires different columns and various solvents are used to elute the mixture through the column. Typical columns include sulfonated polymeric or amino-bonded silica columns. Typical solvents include acetonitrile/water mobile phase. Gas liquid chromatography is a sensitive technique and allows the analysis of sub-nanomolar amounts of carbohydrates. 14 Capillary electrophoresis is a newer technique that yields results in relatively short times and with high efficiency. To achieve electrophoretic separation, the two ends of the capillary are submerged into two separate electrolyte reservoirs that contain a high voltage electrode. The separation is due to the variation of molecular size and electric charge ratios of the sugars within the mixture. This method does not require derivatization of the oligosaccharides and cannot be used to identify and separate oligosaccharides that have the same degree of polymerization, i.e. isomers. Derivatization of oligosaccharides allows for them to be more volatile and therefore more compatible with analysis methods such as mass spectrometry. One common derivative method is hydrolysis followed by chromatographic separation. 22,23 Besides hydrolysis, other common methods used to derivative oligosaccharides are permethylation 24 and peracetylation. 25 Permethylation has been shown to easily determine branching and interglycosidic linkages. It also helps stabilize sialic acid residues in acidic oligosaccharides and in conjunction with matrix-assisted laser desorption ionization (MALDI) has been shown to give more predictable ionization than non-permethylated oligosaccharides. 26 Two common methods for permethylation are the use of dimethyl sulfoxide anion (DMSO - ) to remove protons from the 23

24 analyte and replace them with methyl groups 27 and the addition of methyl iodide to DMSO - which contains powdered sodium hydroxide. This second method effectively replaces protons with a methyl group at both oxygen and nitrogen sites in oligosaccharides. 24 Analysis Methods Once released and separated, the oligosaccharides can then be analyzed individually. One past method for differentiation of isolated and separated carbohydrates is NMR spectroscopy Over the past 25 years advances in NMR have allowed it to become suitable for structural analysis of carbohydrates. 31 Such advances include improvements in instrumentation, pulse sequences, ability to interpret spectra, isotopic labeling of compounds and improvement in molecular modeling. With the advances of technology, the ability and accessibility of these techniques have become faster, better and more accessible. The improved coupling of molecular modeling with NMR has provided the ability to determine primary structure and three-dimensional structures of different biological molecules. 31 While NMR has been used to study carbohydrate structures, including glycosidic linkages of saccharide units, and has developed considerably in recent years, it still has several drawbacks and areas in need of improvement. First, the sample size required for NMR analysis is relatively large. Another major drawback is that data analysis can be complicated and time consuming. Typical 1 NMR spectra can be used to give partial spatial arrangement, but due to the incomplete separation of the proton resonance signals they cannot provide a lot of structural information. Other types of NMR have been used in the past to analyze carbohydrates and include 13 C, 15 N, 17 O, 19 F and 31 P. The resolution and sensitivity of each method varies and therefore different information can be ascertained by using each method. For example, 13 C-NMR can give the information of the anomeric configuration of the carbohydrate residues. It can also provide sequence information of the composite monosaccharides, their sequence and 24

25 the overall conformation of the carbohydrates. Another NMR method that improves the results, but increases the complexity, of data analysis uses 2D- homonuclear correlation types of spectra (2D-COSY) to assign resonances and give further structural information. Although these spectra give more information, they do not provide monosaccharide sequence information because there is an absence of coupling over the glycosidic linkage. For this, nuclear overhauser enhancement spectroscopy (NOESY) or rotating-frame overhauser enhancement spectroscopy (ROESY) may be used. While there is some success with these methods, the linkage is not always identified. 31 Since carbohydrates are inherently flexible, in solution carbohydrates may undergo alternations. Estimation of the solution structure required knowledge of the configuration of the composing monosaccharides. Flexible motions of the whole molecule on a short time scale involve fast vibrations at bonds and angles and on a longer time scale involve changes in the dihedral angles. Therefore changing the relaxation time can help deduce the internal flexibilities of carbohydrates in solution. As one can see, the data required for this type of analysis are extensive and analysis can be extremely time-consuming. Mass spectrometry is another very popular analytical technique that is used for gas-phase analysis of carbohydrates. Several types of mass spectrometers have been used for analysis, including Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), which will be discussed further in chapter 2. Mass spectrometry has been shown to have 3 to 4 orders of magnitude higher sensitivity than NMR. 32 Mass spectrometry is highly sensitivity and can be used in multi-step approaches to determine structural information. In order for analysis with mass spectrometry to be done, one of several ionization methods can be used to introduce the analyte of interest into the mass spectrometer. 25

26 Mass Spectrometry: Ionization Techniques Both hard and soft ionization methods exist. ard ionization methods are ones that result in fragmentation and degradation of the sample during the ionization process, whereas soft methods produce little or no fragmentation during the ionization process. One previous hard ionization method widely applied is electron ionization (EI). In EI a beam of electrons is used to excite and ionize a volatile analyte. A main drawback of EI is fragmentation of the sample before detection. 33 Soft ionization methods are currently preferred since they result in the ionization with molecules of the sample remaining intact. Electrospray ionization (ESI) is the most popular of the soft ionization methods. Several soft ionization techniques have been used in the past for carbohydrate analysis and include fast atom bombardment (FAB), MALDI 37 and ESI. 38,39 In FAB, the analyte is mixed with a liquid matrix and is bombarded under vacuum with a high energy beam of atoms. Fast atom bombardment results in the release of [M+] + or [M-] - ions which can then be analyzed. 40 Analysis with FAB had several constraints including poor ionization of neutral and basic oligosaccharides and restriction of analysis to relatively smaller molecules. While basic oligosaccharides were ionized poorly with FAB, acidic oligosaccharides produced stronger signals in the negative ion mode. 35 When FAB was coupled with FTICR-MS, extensive fragmentation, including cross-ring cleavages was seen. 41,42 While FAB uses a liquid matrix, MALDI uses a crystalline matrix where the analyte of interest is co-crystallized with the solid matrix molecules. A laser is focused onto the matrix and its photon energy is absorbed by the matrix and the analyte of interest is released as charged ions. 43 While analysis with traditional MALDI is possible, analysis of smaller saccharide units is a challenge because most peaks of the typical matrix are present in the range m/z <500, where peaks due to smaller saccharides such as mono-,di- and trisaccharides are also found. Recently, 26

27 use of an acid fullerene matrix instead of the traditional matrix has allowed for disaccharides to be successfully studied with MALDI. 44 This approach needs to be developed more fully and applied to other types of carbohydrates. Electrospray ionization is the least energetic of these three gentle ionization techniques. A primary benefit of ESI ionization is the absence of matrix peaks and therefore ease of analysis for smaller mono-, di- and trisaccharides. 33,45,46 In ESI, a solution of the analyte and solvent is passed through a capillary with a high voltage (2 to 5 kv) applied to it. 47 This process allows for charged droplets to be formed. Once formed these charged droplets can then be transferred (through differential pumping and ion optics) into a mass spectrometer and analyzed by mass spectrometry. Electrospray ionization is versatile when it comes to carbohydrates since it can be used to ionize both basic and acidic oligosaccharides. Since multiply charged ions are formed, and mass spectrometers typically separate based on mass-to-charge ratio, ESI has virtually no limit to the size of the ion that can be analyzed. This dissertation will concentrate on ESI since it was used exclusively in the research to be reported. Fragmentation Methods Since isomers have the same mass and therefore cannot be differentiated by mass spectrometry alone, differences in ion fragmentation can be used to distinguish isomers. To obtain structural information, several fragmentation methods have been used. These methods include electron capture dissociation (ECD), collision induced dissociation (CID) and infrared multiple photon dissociation (IRMPD). Electron capture dissociation uses low energy electrons to induce fragmentation of the saccharide. 6 It results in multiply, positively charged ions that can then be analyzed with a mass spectrometer. Past research has included using ECD to do top-down analysis where a whole protein is sequenced simultaneously. Also, O-glycosylation sites on proteins have been explored 27

28 using ECD. 48 While ECD can be used for proteins and peptides, it has limited application to oligosaccharides. Another dissociation technique that is more applicable to oligosaccharides is CID. In on-resonance or traditional CID, a neutral background gas is pulsed into the cell, analyte ions are accelerated to higher kinetic energies, and collide with the introduced gas. These collisions result in fragmentation of the analyte of interest. 49 Another commonly used CID method in FTICR-MS is sustained off-resonance irradiation collision induced dissociation (SORI-CID). 50 In SORI-CID, ions are excited by an off-resonance frequency, causing their kinetic energies to increase and decrease repeatedly with time, resulting in less-energetic collisions with background molecules over a longer time period than with conventional CID. These collisions can nonetheless result in fragmentation of an isolated ion of interest. Collision induced dissociation of oligosaccharides results in fragments that can be used to determine stereochemistries, linkage position and branching information. 34,51,52 A disadvantage to SORI-CID with respect to identification of oligosaccharide is that since SORI-CID is low energy, cross-ring fragmentations are less likely than the fragmentation of the glycosidic linkage. Also, the ability to control the energies of collisions is limited with CID. Due to the collisions and variance of energy, CID can give different fragmentation than other dissociation methods. One fragmentation method that gives similar and complementary fragments to CID, but allows for finer control of the energy imparted to the system is IRMPD. 53 IRMPD relies on absorption of photons by one vibrational normal mode of trapped ions and the subsequent redistribution of photon energy into other vibrational modes of the ions. This redistribution occurs via intramolecular vibrational relaxation. 54,55 If sufficient photons are absorbed without excessive collisional or radiative relaxation, then the internal energy of the ion increases to a 28

29 level above the dissociation threshold, resulting in fragmentation. One advantage of IRMPD over CID is that the power is only limited by the laser being used. Therefore, use of a tunable laser gives finer control over the power imparted into the system. The theory and history of IRMPD will be discussed in more detail in chapter 3. A systematic nomenclature method has been developed for naming fragments of carbohydrate ions. In this method, the fragments which contain a non-reducing end sugar are labeled with uppercase letters sequentially starting with A, Figure Those fragments that contain the reducing sugar are labeled sequentially with letters from the end of the alphabet (X, Y, Z). Ions formed by cleavage across a ring are A and X ions. The subscripts for these fragments are given by assigning each ring bond a number and then counting clockwise. Charged Ions Since mass spectrometry only detects charged particles, metal ions have become a common way to ionize neutrals and then detect the complexes formed with mass spectrometry. Adduction of an alkali metal ion has been used with FAB, MALDI and ESI in both the positive and negative ion mode For fragmentation of a metal-attached ions two pathways predominate. The first type of fragmentation is loss of the metal ion and the second type is fragmentation of the molecule into smaller charged parts which often retain the metal ion. The fragmentation pathway that occurs depends on the strength of the bonds of the adduction of the metal to the molecule, Figure If the binding energy of the metal ion is less than the dissociation threshold, then loss of the metal will occur. This type of fragmentation is seen when large alkali metals are adducted to molecules. This is because the binding energy of the larger alkali metals ions is lower than that of smaller alkali metal ions. 58 The opposite has been seen with the smaller alkali metal ions. Since their binding energies are larger and thus metal ion dissociation is less likely, the result is 29

30 greater fragmentation of the molecules with the smaller alkali metal ions remaining attached to the fragments. Cancilla et al. found that the relative binding energy for alkali metal ions is Li + >Na + >K + >Rb + >Cs The stronger the binding energy, the more fragmentation that will be seen with IRMPD since it is more likely the molecule will fragment before losing the metal. 6 Xie et al. have compared the ability of CID and IRMPD to fragment alkali-adducted molecules and showed that for smaller ions such as Li + and Na + both dissociation method yielded similar fragments. 62 Specifically, adduction of lithium to saccharides has been studied by ofmeister et al. 60 In this research they determined that the lithium cation interacts with disaccharides through several oxygen sites, including the glycosidic bond. This triple interaction leads to stronger binding and therefore greater fragmentation is seen with IRMPD. The research performed in this dissertation primarily used adduction of lithium ions and analysis in the positive ion mode. In the negative ion mode, Cole & Zhu have shown that chlorinated species can be studied conveniently. 61 Formation of the chlorine adduct has proven successful for species that are polar, neutral molecules or slightly acidic molecules that do not generate negative ions through deprotonation. Therefore, chlorination has been shown to be one easy method for exploring ions in the negative mode when addition of a strong base does not promote deprotonation. While the addition of an appropriate salt can help facilitate the ESI process through producing charged adducts, excessively high salt concentrations can cause background interferences; therefore caution needs to be taken when using salts for the creation of ions. These interferences can lead to signal suppression and the subsequent inability to detect the ions of interest. The ease of the adduction of metals to create ions with oligosaccharides makes their 30

31 use with IRMPD a promising method to differentiate the sugars in both positive and negative ion modes. Objective of This Research Since carbohydrates are biologically important, being able to differentiate both their linkages and anomeric configurations can give valuable information. For this research, FTICR-MS was used in conjunction with IRMPD to distinguish various mono- and disaccharide ions in both the positive and negative ion mode. Fourier transform ion cyclotron resonance mass spectrometry not only gives superior mass resolution and mass accuracy when compared to other types of mass spectrometry, but it also allows for tandem mass spectrometric experiments to be done in the same region of space (within the analyzer cell), thereby eliminating extra instrumentation that is often needed with other mass spectrometers. 63,64 Since IRMPD uses lasers to introduce photons, various lasers have been used in the past including fixed frequency and wavelength-tunable CO 2 lasers and free electron lasers (FELs). 55,70-72 Fixed frequency CO 2 lasers produce photons at one wavelength (10.6 μm), thus the information that can be obtained with them is limited. Free electron lasers, on the other hand, have a large output wavelength range (5 to 250 μm) but these lasers are very expensive and access to beam time is limited. Therefore, a less expensive alternative with at least a (limited) range of wavelengths (9.2 to 10.6 μm) is the tunable CO 2 laser that will be emphasized in this research. The objective of this research was to produce a method for discriminating between various linked and anomeric configurations of mono- and disaccharides. While previous research done by Polfer et al. with irradiation produced by a FEL had shown that the linkages and anomeric configurations could be distinguished by wavelength-dependent ion fragmentation patterns, a 31

32 method to do so in more conventional (i.e. non-fel equipped) laboratories had not been demonstrated. 73,74 In this research the anomeric configuration of mono- and disaccharides was determined by examining the fragmentation patterns produced by IRMPD with a tunable CO 2 laser in both the positive and negative ion modes using FTICR-MS. While past methods have studied the lithiated disaccharides in the positive ion mode with FEL irradiation, the negative mode of mono- and disaccharides has not been explored. Therefore the fragmentation of glucose-containing disaccharides, some of their specific fragment ions and some selected monosaccharides was also examined in the negative ion mode at the Free Electron Laser for Infrared experiments (FELIX) facility. Overview The next chapter will give a description of FTICR-MS. This description will include a history as well as theoretical and practical aspects of FTICR-MS. Chapter 3 will discuss the mechanism and theory of IRMPD. The types of lasers used for IRMPD will also be described in this chapter. Chapter 4 is a detailed description of the procedure and apparatus used to differentiate lithiated monosaccharides with a tunable CO 2 laser at the University of Florida in Dr. John Eyler s laboratory. The results of this study will also be discussed. Chapter 5 will discuss a method to determine both the linkage and anomeric configuration of lithiated glucosecontaining disaccharides in the positive ion mode with a CO 2 laser. Chapter 6 will next describe IRMPD fragmentation of deprotonated and chlorinated disaccharides in the negative ion mode by wavelength-tunable CO 2 laser. A description of the procedure and apparatus used for the fragmentation of deprotonated disaccharides done at the University of Florida in Dr. David Powell s laboratory will also be given. Chapter 7 will give a detailed account of negative monoand disaccharides ions and some of their fragment ions explored at the FELIX facility. Finally, a 32

33 conclusion including a summary of the strengths and weaknesses of this work along with proposed future work will be presented. 33