gas-phase non-covalent complex - from inclusion complexes to single cell

It also explains the mechanism, instrumentation, application of electrospray ionization ESI source and Fourier transform mass spectrometry FIMS.. Asparagine Aspartic acid Aerosol time-of

BY Xin Cong B.S (Nankai University) 1998 M.S (Nankai University) 2001

DISSERTATION Submitted in partial satisfaction of the requirements for the degree of

DOCTOR OF PHILOSOPHY

in Chemistry

in the OFFICE OF GRADUATE STUDIES

of the UNIVERSITY OF CALIFORNIA

DAVIS Approved

Coro QU

“na; é Abed

Committee in Charge

2006

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1

First of all, I would like to express my sincere appreciation to my research director, Professor Carlito B Lebrilla, for all his support and encouragement This work would not have been possible without his direction and guidance In particular, his confidence in me made the BAMS project possible I have learned about American culture from him, as well as research during the whole graduate program He is both a helpful advisor and a humorous friend

I would also like to thank all the members in Dr Lebrilla’s research group I can always rely on them whenever there are problems Especially I would like to thank Mitchell Stone and Dr Gregg Czerwieniec who lead my way into my first project and the mass spectrometry world Dr Yongming Xie, Dr Andreas Franz, Dr Jinhua Zhang and

Dr Scott Russell taught me about mass spectrometry during my early days in the group

My BAMS group, Dr Scott Russell, Dr Gregg Czerwieniec and Richard Seipert, made the days working in machine shop enjoyable Fellow group members Kate Lancaster (lunch buddy!), Caroline Chu, Eric Dodds and Richard Seipert gave me helpful advices

on this dissertation Moreover, I would like to thank to, Dr Brian Clowers, Dr Hyun Joo

An, Crystal Kirmiz, Nicolas Young, Bensheng Li, Milady Ninonuevo (and Caroline, as Walmart Buddies, Yeah!), Erica Mcjimpsey, LaTasha LaMotte and Nannan Tao for their

valuable suggestions and discussion and for the pleasant atmosphere they produced in the lab Special thanks should be given to Michael Sisto and Blaine Hutson from machine shop, and Skipp May from electronic shop Without their help, the BAMS project would still be designs on paper

iii

China and in U.S Their support during the past four years helped me go through the hardest days of graduate study and realize my dream

1V

January 2006 Chemistry

Gas-phase Non-covalent Complex — From Inclusion Complexes to Single Cell

Abstract

Chapter 1 provides an introduction to the significance and methodology of gas- phase non-covalent complex study It also explains the mechanism, instrumentation, application of electrospray ionization (ESI) source and Fourier transform mass spectrometry (FIMS) A home-built 4.71 ESI-FTMS employed in experiments is presented

Chapter 2 presents evidence of the gas-phase zwitterionic amino acids in the cavity of cyclodextrin Zwitterionic amino acids are discovered in a ternary complex with underivatized B-cyclodextrin unlikely with derivatized B-cyclodextrin used in previous reports Ternary complexes are produced after a proton transfer of alkylamine from carboxyl group of amino acids The ternary complexes are the result of zwitterions formation The experimental results and molecular dynamics calculations confirm the existence of the gas-phase zwitterionic amino acids inside of cyclodextrin

Chapter 3 describes the kinetics study on the reactions mentioned in chapter 2 The factors affecting gas-phase zwitterion formation were investigated The rate constants of the reaction were obtained through the kinetic plot The enantioselectivity of

dynamics calculations were performed to confirm the structural assumptions

Chapter 4 gives an introduction to bioaerosol mass spectrometry (BAMS) History and facts of bioterrorism are presented Instrumentation of various bioaerosol mass spectrometers is explained In addition, introduction to themethodology of single cell analysis is provided

Chapter 5 describes the development of a novel bioaerosol Fourier transform mass spectrometer (BAMS-FT) Design of the instrument is supported by SIMION simulation The principle of the instrument is explained in detail Primary results of aerosol particle tracking are provided

VỊ

Asparagine Aspartic acid Aerosol time-of-flight mass spectrometry Magnetic Field

Bioaerosol mass spectrometry Tri-O-methyl B-cyclodextrin Di-O-methyl B-cyclodextrin Cyclodextrin

Capillary electrophoresis Collision induced dissociation Capillary liquid chromatography Consistent valence force field Continuous wave

Cysteine Dalton 2,6-dihydroxybenzoic acid Desorption/Ionization Dipicolinic acid Diode-pumped solid state Electron capture dissociation Ethylenediamine

Electrospray ionization Electron volt

Fast atom bombardment Fourier transform mass spectrometry

Vil

Peak intensity Ion cyclotron resonance Isoleucine

Infrared multiphoton dissociation Liquid chromatography

Leucine Lysine Mass Mass-to-charge ratio Matrix-assisted laser desroption/ionization Methionine

Molecular dynamics Methanol

Maltoheptaose Mass spectrometry Tandem mass spectrometry Nano-scale liquid chromatography Nano-scale electrospray ionization Nuclear magnetic resonance n-propylamine

Polymerase chain reaction Phenylalanine

Photomultiplier tube

Proline

Vill

Quadrupose ion trap Radio frequency Severe acute respiratory syndrome Sodium dodecy! sulfate

Serine Solid phase extraction Threonine

Time-of-flight Tryptophan Transistor-transisitor logic Tyrosine

Velocity Valine Peak-to-peak voltage Cyclotron frequency

1X

Chapter 1

Introduction to the Investigation of Gas-Phase Non-covalent Complexes by Electrospray lonization (ESI) Fourier Transform Mass Spectrometry (FTMS)

Introduction

Cyclodextrin Inclusion Complexes

Electrospray lonization (ESI)

Fourier Transform Mass Spectrometry (FTMS)

Ion Motion Jon Excitation and Detection Instruments

Structural Relationships in Small Molecule Interactions Governing Gas-phase

Enantioselectivity and Zwitterionic Formation

Introduction to Bioterrorism, Bioaerosol Mass Spectrometry

and Single Cell Analysis

Introduction

Bioterrorism

Bioaerosol Mass Spectrometry

Single Cell Analysis

Mass Spectrometer

Introduction

Proposed Instrument

Instrument Design and Construction

Particle Generation, Sampling and Tracking

Introduction to the Investigation of Gas-phase Non-covalent Complexes by Electrospray Ionization (ESI) Fourier Transform Mass Spectrometry (FTMS)

Non-covalent interaction is an important phenomenon in the fields of biology and biochemistry In contrast to covalent bonds, non-covalent bonds are weak interactions

among ions, molecules, and parts of molecules Non-covalent interactions include: charge-

charge interactions, hydrophobic interactions, hydrogen bonding interactions, Van der Waals forces (also known as London dispersion forces), charge-dipole interactions, and dipole-dipole interactions They often work in combination to yield significant effects The mobility and instability of non-covalent bonds make them the most common interactions between biological macromolecules.’ A good example is the intramolecular non-covalent forces that are responsible for the secondary and tertiary structure of proteins and their functions in organisms The non-covalent interactions between two or more proteins are the basis for quaternary protein structures In pharmacology, most drugs

interact non-covalently with biomolecules, such as proteins and RNAs, at specific sites

and/or in specific conformations “Sẻ

Many traditional methods, including fluorescence, phosphorescence, NMR and X-ray crystallography, have been applied to the investigation of non-covalent complexes For example, 1H NMR spectroscopy has been proved to be very useful in structure elucidation of inclusion complexes * > Other spectroscopy techniques, such

as, Rydberg electron transfer spectroscopy, photoelectron spectroscopy and anion infrared spectroscopy, ®*13 are also employed to investigate the structural dependence of electron transfer to non-covalent polar complexes, which is fundamental in many biological processes such as photosynthesis

For example, the NMR spectra of biological macromolecules are difficult and time-

Ũ : 4

consuming to interpret * '* F3 X-ray crystallography provides in-depth coordinate information of non-covalent complexes However, it requires good crystalization Growing crystal is a delicate and lengthy process, and fine crystal is not a guaranteed result Even though satisfactory crystal is obtained, the conformation of the complex may not be the same as in the solution '°

Mass spectrometry, with recent progress of ionization methods, provides an

17, 18

alternative approach for the study of non-covalent complexes Early on, the application of mass spectrometry was limited on characterizing small molecules The analysis of biological macromolecules was restricted because most of them are too labile to survive harsh ionization processes In the 1980’s, novel soft ionization

19 20, 21

techniques such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESD 22, 23 was developed and applied to the study of large biomolecules Upon the last two decades, mass spectrometry has brought the non- covalent complex study to a new level 23-26 Mass spectrometry is not only an effective means for structure elucidation of peptides, proteins and other biomolecules, 7” ** but also a powerful tool in investigating the non-covalent interactions of these biological

macromolecules 772 '7 !® 33 In this research, our goal is to study gas-phase non- covalent complexes utilizing mass spectrometry and to develop new instrumentation for this purpose

Cyclodextrins (CDs) are cyclic oligomers of œ-D-glucose with œ-1,4 glycosidic linkage They have a truncated-cone shape with a cavity of 7.9 A in depth They were first isolated from starch by growing a culture of Bacillus amylobactor on a starch- containing medium ** The most commonly used enzyme to yield CD is Bacillus macerans which was first successfully isolated by Schardinger °>-3° The enzymes are not very specific; the oligomer generated by them has a range of glucose units The

most abundant ones contain 6, 7 or 8 monomers, which are so-called a, B andy -CDs

The number of glucose subunits determines the cavity size of the CD cone shape The

wide and narrow rims’ diameters of the CDs are 5.3 A and 4.7A for the o-CD, 6.5 A

and 6.0 A for the B-CD, and 8.3 A and 7.5 A for the y-CD Figure 1.1 shows the structure of B-cyclodextrin The secondary hydroxyl groups of C2 and C3 on the glucose compose the wider rim, and the primary hydroxyl groups on C6 make up the narrow rim The interior of the cavity is non-polar compared to the rims because the non-bonding electrons on the glycosidic oxygen bridge point to the inside of the cavity Moreover, the hydroxyl groups on the rims of CDs can be easily derivatized to improve their solubility and functionality Replacing the hydroxy! groups of the bottom and top rim of the torus with methoxy groups breaks intra-molecular hydrogen bonds, making the cyclodextrin much more water-soluble Inclusion complexes with methylated cyclodextrins have greater stability than complexes of the native form

various compounds such as hydrocarbons, aliphatic alcohols, diols, amines, acids, cyclohexane derivatives, amino acids, oligopeptides, sugars, phenols, aromatic amines, azo compounds, naphthalene derivatives, other aromatic compounds and various drugs

5 37

In solution, the major driving force behind the inclusion complexes is non-

covalent interactions, including van der Waals and hydrophobic interactions, with

hydrogen-bonding and steric effects also playing a role 3837 Th aqueous solution, water molecules are located in the CD cavity The CD complexes with water molecules are energetically unfavorable because of the relatively non-polar interior of CD Once a non-polar moiety of a guest enters the cavity, the water molecules are dispalced from the cavity, and the guest is encapsulated inside the cavity Complex formation establishes an energetically favorable “hydrophobic” interaction in solution Hydrogen bonding or van der Waals forces alone are too weak to produce complex formation When two or more molecules are in a specific spatial arrangement that leads to several specific interactions, these molecules may achieve the stability that is commensurable with the magnitude of a covalent bond “

Cyclodextrin inclusion complexes possess properties such as enhanced guest solubility, controlled release of volatile guest and protection for labile guest > 40 which makes it well suited for applications in the field of pharmaceutical and food sciences

For example, in the food industry, CD is utilized to stabilize food flavors, volatile

molecules and dyes “1” CDs are also employed in separation techniques like high-

43 44-46

performance liquid chromatography, capillary electrophoresis, “7 and gas

include the use of cyclodextrins for drug encapsulation to increase bioavailability of drugs °0 51 52 Th addition, CDs have been found to be a practical tool in biomimetic reactions and molecular recognition 93-60

One of the most common applications of CDs is to achieve chiral recognition ø

In separation science, they are widely used as chiral stationary phase in chromatography 62-70 They have also been used in chromatography as a mobile phase additive.”’ Quantification of enantiomer mixtures is accomplished by employing CDs

Chapter 2 and 3 will discuss the formation of gas-phase amino acid zwitterion formation in the cavity of CDs and the enantioselectivity of the reaction observed in an electrospray ionization Fourier transform mass spectrometer (ESI-FTMS)

Electrospray Ionization (ESI)

Various ionization methods were invented to ionize the sample and send the ions

to the mass analyzer Early ion sources included electron impact and chemical

Ionization of biological macromolecules is problematic because they are insufficiently volatile and/or are thermal stable to permit volatilization prior to ionization The

invention of fast atomic bombardment ionization (FAB) was one of the earlier attempts

to solve the problem of involatility 91 82 With FAB ionization, samples in a condensed state, often in a glycerol solution or “matrix”, are ionized by bombardment with energetic xenon or argon atoms °° This method had restricted application because of experimental difficulties and limited range of molecular masses accessible to it * The thermospray method was another attempt °° 98 In the thermospray method, solvent evaporation is realized by radiative heating of a fine spray of droplets However, this technique was limited with respect to the volatility and polarity of the analytes

Electrospray is one of the two relatively new ionization methods, which are widely utilized to solve biological problems now Like the other extensively used

ionization technique, matrix-assisted laser desorption/ionization (MALDI), 0 ESI

greatly facilitates mass spectrometric analysis of intact ions generated from condensed- phase macromolecules like biopolymers (peptides, oligonucleotides, oligosaccharides) and synthetic polymers

The concept of electrospray has been around for more than a century Chapman was more specific to carry out experiments with it ?” In the late 1960’s, Dole er al developed the electrospray ionization method for mass spectrometry They generated gas-phase ions of macromolecules by electrospraying them from solution °*”’ In the 1980’s, electrospray ionization mass spectrometry (ESI-MS) became a modern-day analytical technique through the effort of Fenn and co-workers They developed

electrospray as a true interface and coupled it to a quadrupole mass spectrometer With the ESI-MS, they reported the detection of poly(ethylene glycol) with average molecular weights up to 17,500 kD and naturally occurring biopolymer, e.g., proteins

up to 40,000 kD by electrospray '°°?

Since the invention of new ionization techniques including MALDI and ESI, mass spectrometry has grown to be one of the leading tools in biochemistry and related fields The soft ionization makes it possible to generate intact gas-phase ions directly from biological buffer solutions, close to physiological conditions that help preserve the structure and activity of the labile molecules The internal energy transferred to the biological macromolecule during the ionization process is sufficiently low to produce intact molecules

Another advantage of ESI is that it often creates a distribution of multiply charged ions corresponding to (M + nm,)"", where M is the molecular weight, m, is the

adduct ion, and n the number of ionic charges Therefore, the m/z value of ions of large

biological molecules can be made relatively low if the number of charges is large This plus is especially beneficial to protein and peptide research because the m/z values of the analytes are significantly lowered by multiply charged basic amino acid residues 103 However, when dealing with unknown mixtures, multiply charged ions complicate the interpretation of mass spectra Fenn and others developed deconvolution algorithms

104

that aided in interpreting mass spectra of multiply charged ions For a single component with molecular mass M:

in which K,, is the location of the peak on the m/z scale For any two peaks separated by

j charges the adduct ion mass can be determined from

in which the sum is over all the peaks and p is the number of peaks

In addition, the development of microspray and nanospray techniques, which offer high sensitivities and low limits of detection, has provided the capability of characterizing biologically important molecules available only in very small! quantities Sensitivity has been pushed into the low picomolar and attomolar levels 26101 Hệ Hà

ESI also offers a facile interface betweengmnass spectrometry and separation techniques such as liquid chromatography (LC) and capillary electrophoresis (CE) số 195 The invention of microchip techniques maximize the capability of MS as well as reduce the required sample amount, analysis time and the costs '°° For proteomics study, not only sample separation, but also other pretreatment steps such as enzymatic digestion,

107 108 and preconcentration, 109 110 can be carried out in the microfluidic devices coupled to ESI

The ESI technique provides simultaneously solution introduction and ionization

of highly polar and involatile compounds into the mass spectrometer The principle of the ESI process is the transfer of analytes, which are normally ionized in condensed phase, into the gas phase as isolated species The mechanism of ESI is generally divided into three parts: droplet formation, droplet shrinkage and gaseous ion

formation.''*

As shown in Figure 1.2, droplet formation occurs by the application of a high voltage on the electrospray capillary needle In conventional electrospray the analyte solution flow rates are set between 1-5 ml/hr The metal needle is of 0.2mm o.d and 0.1mm ¡.d., and 2-5 kV is applied to it For microspray, only about 1-2 kV are required

to produce droplets with the flow rate at 8-18 ul/hr The needle is usually made of fused silica with a 150 um o.d and 50-25 um i.d In this work microspray was used The inlet to the mass spectrometer is always fixed at a certain distance away from the needle and set at a lower potential (often ground) producing an electric potential The solution

is transported to the tip of the electrospray needle and experiences a high electric field For positive ion mode, a positive voltage is applied to the tip Positively charged ions

accumulate at the surface of the solution, and then are drawn into a downfield region

forming Taylor cone.''° From the tip of the Taylor cone a narrow jet of liquid emerges After a path of about 1-2 mm, the jet spreads into a fine mist of droplets Droplets from

the Taylor cone are emitted towards the mass spectrometer inlet (the counter electrode)

It should be noted that the Taylor cone is preferentially enriched with solvated ions of

Figure 1.2 Illustration of electrospray process (Adapted from P Kebarle and L Tang,

Anal, Chem., 1993, 65, 972A.)

similar charge to the applied electric field Although some ions are initially present in solution, electrophoretic charging is also responsible for the production of charged species.''° The charged droplets are accelerated towards the mass spectrometer inlet by the voltage difference between the tip of the needle and the counter electrode The droplets are also trapped in the inward airflow generated by differential pumping and are directed inside the capillary Droplet diameter is influenced by parameters including applied potential, flow rate and solvent properties Under normal conditions

the droplets are considered monodisperse, i.e., they have a narrow distribution of sizes

The typical radius of such droplets is about 1.5 um at flow rates of about 5 ul/min and total electrolyte concentrations of 10° M As the droplets are transported to the MS inlet they shrink in size due to the evaporation of solvent molecules Some mass spectrometers employ a heated capillary to accomplish the evaporation process more efficiently The heated capillary greatly helps desolvated ions to be guided through the electrostatic lenses with high efficiency

Droplets keep shrinking in size by solvent evaporation until they reach a limit where the electrostatic Coulomb repulsion exceeds the surface tension This condition

is called the Rayleigh stability limit At this point the droplets become unstable and undergo fission into smaller droplets The Rayleigh limit gives the condition where the charge of the molecule becomes just sufficient to overcome the surface tension that holds the droplet together There has been some debate as to the kind of fission the droplets undergo It is now generally accepted that the droplets undergo “uneven” fission as opposed to “even” fission where the molecule breaks into two or more droplets of similar or equal size Uneven fission will lead to the formation of smaller

droplets, and eventually to single gas phase ions faster than even fission compared to the flight time of the droplets

The last step in the electrospray process involves the formation of gas phase ions from the solution There are two mechanisms that are now generally accepted Field Evaporation is one mechanism proposed by Iribarne and Thomson.'!” '!® Ton evaporation (emission) is envisaged to occur from small, highly charged droplets The driving force is the repulsion between the charged ions and the other charged species in the droplet The droplet approaches the Rayleigh limit but does not undergo fission A charged part of the molecule penetrates the surface of the droplet The Coulombic repulsion between the surface of the droplet and this part of the molecule expels the entire molecule out of droplet

The second mechanism was proposed by Dole and later supported by the work

of Réllgen.”® The highly charged droplets produced by the electrospray process shrink via evaporation of the solvent Repetitive Coulomb explosions generate nanometer- sized droplets, each containing only one analyte macromolecule The solvent molecules evaporate leaving the macromolecules as ions

In the first model, the macromolecule plays an active role during the ionization process, while, in the latter, it plays a passive one The mechanism proposed by Dole is now the generally accepted mechanism But it is possible that a combination of both may exist during the ionization process and with one dominating over the other at various times The ion desolvation process effectively cools the ions yielding low internal energy ions

The ESI source can be coupled to any MS detectors In our application, it is applied to a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, which provides highly sensitivity, high resolution and high mass accuracy

Fourier Transform Mass Spectrometry (FTMS)

In 1930, E O Lawrence developed the first cyclotron particle accelerator, for the study of nuclear physics.'” He realized that the equations of motion of a charged particle present in a uniform magnetic field predicted a mass dependent period of revolution These equations of motion also predicted that the particle could be accelerated if its motion was in resonance with an applied radiofrequency field These discoveries form the basic principles of the mass spectrometric technique that derives its name from such a motion, ion cyclotron resonance mass spectrometry, ICR-MS The first application of the cyclotron principle to obtain mass assignments of charged particles was realized by Sommer, Thomas and Hipple They constructed the first ICR-

MS in 1950, calling it the “omegatron”.'* This instrument was used to measure the

mass to charge (m/z) ratio of the proton These first generation ICR mass spectrometers

utilized a rectangular, three-region drift cell for mass analysis Here, ion formation and

detection were accomplished in different regions of the cell With the use of a voltage differential, ions formed in the source were made to drift to the resonance region, where they were mass detected.!*? In 1970 McIver introduced the first ICR cell in which both ion formation and detection events occur at the same location in the cell.'*° This design differs first in that the trapped ion analyzer cell uses a static electric field to trap ions along axis parallel to the magnetic field lines Additionally, the trapped ion analyzer

cell relies entirely on a pulsed mode of operation while the drift cell configuration used

a continuous current of ionizing electrons and continuous drift of ions through the length of the cell Four years later, Comisarow and Marshall combined the technology

of pulsed ICR-MS with Fourier transform techniques.'*’ The FT-ICR technique has the advantage of being able to acquire a mass spectrum orders of magnitude faster than the previous conventional ICR The FTMS instrument is essentially a multichannel mass spectrometer, where all the ions are detected simultaneously, while standard ICR mass spectrometer was a single channel, scanning instrument

The application of FTMS in analytical chemistry offers several distinct advantages over other mass spectrometric methods One important feature is its ability

to achieve very high mass resolution For instance, a typical mass resolution of 100,000(FWHM) for m/z 1000 can be obtained for experiments performed at low pressures (10° torr) with a 5 Tesla magnetic field.'*° The mass resolution associated with FTMS is proportional to the observation time and magnetic field strength, and inversely proportional to the mass of the ion FTMS also offers the ability to determine the mass of an analyte accurately This is the result of the fact that frequencies can be measured with great precision allowing accuracies of parts-per-trillion to be obtained.'”° Another benefit of FTMS is that the detection event is nondestructive in nature so that it can be repeated many times to improve the signal-to-noise ratio The signal intensity increases with the square root of the number of scans Following detection, the voltage

of a trapping electrode is biased to remove all ions from cell However, if after detection the ions are allowed to relax to the cell center by collision with a background gas, they can be re-excited and detected again.'”" !2 Thịs provides a very sensitive

means to mass analysis with important applications to biological analyses To understand the reason for the advantages of FTMS, an introduction of FTMS principle

5 V) to the trapping plates Either positive or negative ions can be trapped within the cell by changing the polarity of the trapping plates The basic equation predicting the motion of the ions trapped in this manner is derived from the equation showing the

inward and outward forces on the ion:

where F, E, v, B and q are the force, electric field, velocity, magnetic field, and q is the

charge of the ion, respectively The equation describes the force exerted on a charged particle by both the electric and magnetic fields

The solution for the frequency of revolution (cyclotron frequency) is produced

œ =qB/m (1.5)

where W is the cyclotron frequency, q is the charge, B is the magnetic field strength, and

m is the mass This cyclotron motion constrains the ions to cyclotron in the x-y plane, as shown in Figure 1.3

Figure 1.3 [on motion in a magnetic field The ion moving perpendicular to a magnetic field is constrained to a cyclotron motion with a characteristic frequency

From this equation it is apparent that the cyclotron frequency is proportional to the magnetic field strength, B, and inversely proportional to the mass-to-charge ratio, m/q of the ions Cyclotron frequencies fall in the range of 5 kHz to 5 MHz The mass- to-charge ratio is determined by measuring the cyclotron frequency Although Equation 1.5 is accepted as the general expression associated with FT-ICR MS, it does

not account for all the forces imposed upon an ion within the cell, nor does it

completely describe the cyclotron frequency An important feature in FTMS is that the cyclotron frequency is independent of the ion’s kinetic energy allowing for very precise

measurement of the cyclotron frequency This results in high resolution and high mass accuracy in FTMS

The magnetic field exerts a constraining force in the x and y directions, but the ion experiences no force parallel to the field (z direction) and is free to drift out of the cell along this direction Ion trapping along the z-axis is accomplished by applying and electrostatic potential to the two plates on the ends of the cell Introduction of a trapping potential introduces an oscillating motion known as the tapping frequency The trapping frequency is described by Equation 1.6:

ion motion known the magnetron motion, which also has a characteristic frequency, fi,

is given by Equation 1.7

The magnetron motion can be viewed as a slow precession of the guiding center

of the cyclotron orbit along an isopotential contour Magnetron motion results from an interaction of the force produced by the magnetic field of an ion with the radially outward-directed electric field This causes a circular drift to the guiding center of the cyclotron motion around the center of the cell This motion has a: much lower frequency than cyclotron frequency, on the order of 1-100 Hz The magnetron motion serves no analytical purpose Loss of ions from the ICR cell is in part due to the conversion of the cyclotron motion into magnetron motion as the ions lose kinetic energy during collisions with neutral molecules Thus, the efficient trapping of ions generally requires very low pressures (high vacuum pressures of 107 torr or lower) that

minimize the number of collisions

Ion Excitation and Detection

As shown in Figure 1.4, after ions are trapped in the analyzer cell, the ion cloud exhibits incoherent cyclotron motion with a small cyclotron radius (on the order of 100 um) compared to the cell dimensions and is unable to induce an image current Excitation of ions into larger, coherent cyclotron orbits occurs through resonant absorption of an RF waveform of frequency equal to the ion’s cyclotron frequency and can be accomplished by several techniques The earliest type of excitation method is the frequency sweep or chirp, involving a rapid scan over a frequency range of constant amplitude !* Impulse excitation uses a short (Sus) high amplitude pulse in which ions

of all masses are excited instantaneously, making this a non-mass selective technique '”

SWIFT (stored waveform inverse Fourier transform), !*° is a method in whereby the

excitation waveform is defined in the frequency domain Using an inverse Fourier transform, the excitation signal is converted to a time domain waveform and applied to the transmitter plates of ICR cell This results in a nearly flat frequency spectrum and the ability to precisely define which ion or group of ions is to be excited

at large orbit radius at

the same frequency

Figure 1.4 Ion excitation Applying a certain RF field accelerates the ions to larger orbit radius (Adapted from http://ionspec.com/FTMS %20Tutorial/tutorial;fs.htm)

All ions with the same mass-to-charge ratio are excited coherently and move in phase The coherent motion of the ions induces an image current on the receiver plates

Ion detection is achieved by measuring this image current through the receiver plates in

a parallel RC type circuit This arrangement converts the image current (time domain signal) into a voltage that is amplified, digitized and subjected to a fast Fourier transform algorithm (FFT) and is transformed to a frequency-domain spectrum From the frequency domain, the mass to charge ratio (m/z) of the ions are calculated and presented in a mass spectrum with their corresponding relative abundances

Another advantage of FTMS is that ion detection is non-destructive, and ions

can be re-measured to increase sensitivity or the signal to noise ratio After the detection event ions can be relaxed back to the center of the cell by colliding with background neutral gas These ions can be re-excited and re-measured Unlike sector instruments where the specific events in tandem mass spectrometry (MS") are spatially separated, in FTMS the events are separated only in time Thus, tandem mass spectrometry can easily

be performed in the ICR cell by applying specific pulse sequences

Though there is considerable variety in the design of FTMS instruments, they all contain four basic components - a magnet, an analyzer cell (ICR cell), an ultrahigh

vacuum system, and a data system In addition, the ion source can be either located

inside the homogenous region of the magnetic field, or, it can be external to the high magnetic field of the analyzer region

FIMS magnets are generally superconducting magnets, though permanent or electromagnetic magnets can be used But, the high magnetic fields and homogeneity of superconducting magnets makes them superior for FTMS instruments High magnetic field and homogeneity improve mass range, resolution and sensitivity of the instrument Commercial superconducting magnet field strength currently vary from 3 to 12 Tesla

The central component of the FTMS 1s the analyzer cell (ICR cell) Here ions are trapped, excited, and detected The ICR cell is located in the homogeneous region of the magnetic field Many types of cell configurations have been designed, from cubic - one of the first designs- to cylindrical geometries Basic components of the ICR cell are the six electrodes: The two perpendicular to the magnetic field lines are called trapping plate A dc voltage applied to them trap ions from drifting away in the z-direction Four plates lie parallel to the magnetic field One opposite pair called the excitation plates, have a dipolar radio frequency (RF) electric field applied to them to excite the trapped ions The other pair of opposite plates are the detection plates The image current created by the coherent pack of analyte ions flows in the circuit that connects these two plates

Since the performance of the FTMS is more sensitive to pressure, ultrahigh vacuum conditions are essential to achieve high resolution and mass accuracy In FTMS, ions are accelerated to very high velocities and travel a long distance (in most cases, in the order of 10° meters) prior to detection Thus, collision probability is greatly increased In contrast, ions travel only a few meters in most sector, quadrupole, and TOF instruments To achieve these low pressures, cryogenic or turbo molecular pumps are used To obtain the frequencies of ions accurately, the transient must last as long as possible The length of the transient depends on the pressure inside the analyzer cell At high pressure, collisions in the analyzer cell are more frequent resulting in ion

lost, therefore, sensitivity and resolution are hindered Under high vacuum conditions

the number of trapped ions lost prior to detection is much less, thereby improving the signal External ionization sources like ESI or atmospheric pressure ionization, API,

require several stages of pumping between the source and the analyzer cell The configuration of the instrument used in this work requires such differential pumping stages and may includes two mechanical pumps, followed by a turbo-drag (backed by a third mechanical pump), which are used to bring pressure down for the ion entrance in the mass spectrometer Low pressures on the order of 10° torr in the ion guiding quadrupole region along with the analyzer cell achieved via cryogenic pumping

The data system used in FTMS includes a computer, an analog-digital-converter with large buffer memory capacity, a frequency synthesizer, and a delay pulse generator The ions are manipulated and excited using the frequency synthesizer and the delay pulse generator Specific pulse sequences are loaded (or designed) in the data system to accomplish the isolation and/or ejection, excitation and detection of the selected ions The image current detected is amplified and digitized by the analog-to- digital converter The stored transient (time domain data) is Fourier transformed into the frequency domain and the data is presented as a plot of relative intensities versus the mass to charge ratio (m/z)

Instruments

The experiments were performed with a home-built external electrospray ion source Fourier transform mass spectrometer equipped with a 4.7 T superconducting

magnet (Cryomagnetics Inc., Oak Ridge, Tennessee) A schematic of the ion source is

shown in Figure 1.5 For electrospray ionization, a sample solution was introduced via

a syringe pumps with a 10 -15 uL/hr flow rate The microspray tips were home built from silica tubing with an O.D of 150 um and an LD of 25 um The solution was electrosprayed by applying a voltage of 1.5-2.5 kV at the liquid junction on the base of

the microspray tip To avoid contamination between runs, the syringe, sample, sample line and the electrospray tip were disassembled and cleaned with solvent The stainless steel sample line was flushed with several milliliters of solvent The electrospray tip was also replaced with a new unused tip The spray was forced through a stainless steel capillary whose temperature range was maintained at 170 ~ 200 °C for desolvation The capillary was pointed toward two skimmers The capillary and the first skimmer were accommodated in the first chamber that was maintained at 3~5 mTorr by mechanical pumps

The first skimmer opened into a second chamber that was maintained at a pressure of around 10° Torr by a turbo/drag pump After the two skimmers, the ions were focused by two lenses as described in Figure 1.5 The ions entered the quadrupole rods through a shutter Quadrupole rods were utilized as an ion guide and extended through two differentially pumped regions ending at the analyzer cell Two cryopumps maintained the pressure at 4 ~ 8 x 10° Torr in the analyzer cell

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