Instrumentation and Development of a Mass Spectrometry System for the Study of Gas-Phase Biomolecular Ion Reactions Ziqing Lin Purdue University

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INSTRUMENTATION AND DEVELOPMENT OF A MASS SPECTROMETRY SYSTEM FOR THE STUDY OF GAS-PHASE BIOMOLECULAR ION REACTIONS

A Dissertation

Submitted to the Faculty

Purdue University

Ziqing Lin

In Partial Fulfillment of the

Requirements for the Degree

Doctor of Philosophy

May 2015

Purdue University

West Lafayette, Indiana ii

ACKNOWLEDGEMENTS

I am deeply thankful to my PhD advisor, Prof. Zheng Ouyang. He is more than a mentor or supervisor, but a kind friend, giving me a fantastic PhD experience at Purdue.

His passion, courage and extraordinary vision in scientific research makes him an outstanding scientist and engineer. Prof. Ouyang works hard day and night, while on the other side providing a free and comfortable environment for me and other colleagues in the group to do research and raise opinions without pressure. These precious characteristics no doubt affect me in my professional life. When we first met in Tsinghua, he told me that PhD life is a best opportunity to test our boundary of capabilities. I learnt a lot during my PhD study, not only in terms of technical knowledge but the determination and belief in solving a problem. Five years is not a short period, I truly

appreciate his supervision and encouragement for me to explore the scientific world and ii

myself. It is my honor to know Zheng as a person and have the opportunity to work with each other for both research and teaching experiences. I sincerely wish him good luck for his future endeavors.

I would also like to extend my thanks to Prof. Yu Xia in Department of Chemistry. I received a lot of valuable suggestions and comments from her for gas-phase ion chemistry. Prof. Xia is very sophisticated and precise about her scientific findings. Her dedication and persistence shows me how one could possibly be devoted to her career.

Moreover, Dr. Xia has a charismatic personality and always welcomes discussions at any

iii time. She also allowed me to use the nanospray tip puller and the commercial mass spectrometer in her laboratory, which was a great favor during my PhD study.

It is my privilege to have Prof. Kinam Park and Prof. Corey Neu in my thesis committee, who are always trying to do everything to help. Prof. R. Graham Cooks, although not in my committee, has offered tremendous guidance and support in different perspectives of my graduate studies. I would also like to thank Prof. Mingji Dai, Dr.

Yang Yang from Purdue University, Prof. František Tureček from University of

Washington, Prof. Barney Ellison, Prof. Veronica M. Bierbaum from University of

Colorado, Prof. Michael L. Gross from Washington University, and Dr. Amber L. Russell from Badger Technical Services for their kind help in organic synthesis, theoretical calculation, and tips for the pyrolysis nozzle.

I am indebted to all the group members and alumni in Prof. Ouyang, Xia and Cooks’ research group. It has been a wonderful experience to have such a close relationship with so many people. Dr. Tsung-Chi Chen, Dr. Wei Xu, Chien-Hsun Chen and Linfan Li are

the colleagues who helped me to build my instrument. Jason Duncan was a technician in iii

Cooks’ group, always delivering solid supports when I asked for different electronic controls to achieve varies functions. Lei Tan is a close collaborator in Xia’s group with whom I worked together on gas-phase ion chemistry. Besides, it is always inspiring and pleasant to discuss with group members such as Dr. He Wang, Dr. Qian Yang, Dr.

Sandilya Garimella, Dr. Xiaoyu Zhou, Xiao Wang, Yue Ren, Melodie Du, Yuan Su, etc.

Without all your help, I would not have been able to finish my PhD program.

I would also like to take the time to acknowledge my parents who have been supporting me mentally and financially throughout my student life in China and my PhD

iv abroad. They have been extremely patient and understanding and I would like to thank them for all the amazing opportunities they have given me over the years. Last but not least, I owe my deepest gratitude to my lovely girlfriend and dance partner Esther Foo, who shares with me tears and laughter, sadness and joy in my life and for being supportive throughout this journey. Having blessed with a strong memory where I could recall minute details in my everyday life, I’m glad that I would have the chance to remember all the wonderful moments in my PhD life, and for this, I am eternally grateful.

TABLE OF CONTENTS

Page LIST OF TABLES ...... vii LIST OF FIGURES ...... viii ABBREVIATIONS ...... xv ABSTRACT…...... xviii CHAPTER 1. INTRODUCTION ...... 1 1.1 Gas-phase biomolecular ion reactions and mass spectrometry instrumentations ...... 1 1.1.1 Tandem mass spectrometry in gas-phase ion reactions ...... 5 1.1.2 Gas-phase biomolecular ion/radical reactions at atmospheric pressure 7 1.1.3 Instrumentations for gas-phase ion reactions in vacuum ...... 13 1.2 Conclusion ...... 38 1.3 References ...... 40

CHAPTER 2. INSTRUMENTATION AND CHARACTERIZATION OF A HOME- BUILT DAPI-RIT-DAPI MASS SPECTROMETER FOR GAS-PHASE ION/MOLECULE AND ION/ION REACTIONS ...... 50 2.1 Introduction ...... 50 2.2 Instrumentation ...... 52 2.3 Materials ...... 54 2.4 Results and discussion ...... 54 2.4.1 System configuration ...... 54 2.4.2 Pressure effect on gas-phase ion reactions ...... 59 2.4.3 Dynamic gas flow effect on gas-phase ion reactions ...... 62 2.5 Conclusion ...... 69

Page 2.6 References ...... 70

CHAPTER 3. Gas-Phase Reactions of C3H2 with Protonated Alkyl Amines: formation of a c-n covalent bond ...... 73 3.1 Introduction ...... 73 3.2 Instrumentation ...... 74 3.3 Materials ...... 78 3.4 Results and discussion ...... 79 3.4.1 Carbene reaction with protonated alkylamines ...... 79 3.4.2 Theoretical calculation of the reaction mechanism ...... 83 3.4.3 Experimental evidences of the reaction mechanism ...... 86 3.5 Conclusion ...... 91 3.6 Appendix ...... 92 3.7 References ...... 106

CHAPTER 4. Gas-Phase c-C3H2 carbene reactions with biomolecular ions ...... 109 4.1 Introduction ...... 109 4.2 Experimental section ...... 110 4.3 Results and discussion ...... 110 4.3.1 Nucleobases and nucleosides ...... 110

4.3.2 Amino acids, peptides, proteins, and lipids ...... 117 vi

4.4 Conclusion ...... 131 4.5 References ...... 132 VITA…………...... 134 PUBLICATIONS ...... 136

vii

LIST OF TABLES

Table ...... Page Table 1.1 Tandem mass spectrometry dissociation techniques in proteomics ...... 3 Table 3.1 Ammonium reactions with various radical precursors ...... 92 Table 3.2 MS3-MS5 CID spectra of selected reaction products ...... 97 Table 3.3 GDEI spectra of radical precursors ...... 101 + Table 3.4 Ion/molecule reactions of c-C3H3 and neutral amines ...... 104 Table 4.1 Name, structure, and gas-phase basicity3 (GB) of nucleobases and nucleosides4 ...... 111

Table 4.2 Ion/carbene reactions of nucleobases, nucleosides and c-C3H2 ...... 115

Table 4.3 Ion/carbene reactions of amino acids, peptides, protein and c-C3H2 ...... 126

vii

viii

LIST OF FIGURES

Figure ...... Page Figure 1.1 Proton-transfer reaction in the gas-phase vs. the aqueous phase ...... 2 Figure 1.2 Tandem mass spectrometry in space vs. in time ...... 5 Figure 1.3 Radical reactions in the nanoESI plume and bulk solution using low temperature plasma (LTP)35 or ultraviolet (UV) lamp36...... 7 Figure 1.4 Reactions of protonated disulfide peptide and oxygen-centered radicals5 to

form thiyl (-S•), sulfinyl (-SO•), and perthiyl (-SS•) radical ions in nanoESI

plume: (a) peptide chain CLPTR of HMAC-CLPTR, and (b) chain AGCK of AGC(TFTSC)K.42 ...... 9

Figure 1.5 Intramolecular cysteine sulfinyl radical (SO•Cys) transfer with (a) a free

thiol43 and (b) a disulfide bond44 ...... 10 Figure 1.6 Coupling radical reactions in the ESI plume to cleave the disulfide bonds, with

a subsequent MS2 scan to improve sequence information of disulfide viii

peptides5, 35 ...... 11

Figure 1.7 Scheme of Paternò–Büchi (PB) reaction between ketone/aldehyde and olefin

together with possible retro P-B reactions.52 ...... 12 Figure 1.8 On-line coupling of P-B reactions of oleic acid and acetone with negative MS. MS1 spectra of oleic acid (a) before P-B reaction (b) after P-B reaction; MS2 CID of the P-B reaction products at (c) m/z 339.3; and (d) structures of the diagnostics ions (structures 3 and 5). The structures explain the origin of the 26-Da mass difference.52 ...... 13 Figure 1.9 Schematic of mass-analyzed ion-kinetic-energy spectrometry (MIKES) with collision gas for chemical mixture analysis6a ...... 15

Figure ...... Page Figure 1.10 Schematic of tandem time of flight (TOF-TOF) mass spectrometer56 ...... 16 Figure 1.11 Schematic of penta-quadrupole (QqQqQ) mass spectrometer59 ...... 17 Figure 1.12 Symbols and names given to QqQqQ MS experiments with 0, 1, 2, and 3 dimensions. A fixed mass (filled circle) or variable masses (open circle) can be chosen for reactions.61 ...... 18 Figure 1.13 (a) Penta-quadrupole three-dimensional triple-stage (MS3) familial spectrum for 3-octanone ions acquired by selecting ions of m/z 43 as the intermediates. The angled Q1 axis displays the precursors of m/z 43 ions, whereas the horizontal Q5 axis displays the ion/molecule products of these CID-generated m/z 43 ions arising from reactions with isoprene. Triple-stage sequential precursor spectra extracted from the 3D familial spectrum by selection in Q5 of the final products of (b) m/z 81 and (c) m/z 111.63 ...... 19 Figure 1.14 Schematic of flowing-afterglow selected-ion flow-tube (FA-SIFT) coupled with a radical source for ion/radical reactions69 ...... 21 Figure 1.15 Schematic of a basic ICR cell, consisting of two trapping plates (front and back), two opposite excitation plates (right and left), and two detection plates (top and bottom). Ions enter the cell through the front trapping plate. Mass spectrum generated by fast Fourier transformation.74 ...... 23

Figure 1.16 Schematic of the FT-ICR instrument, including the LIAD probe.78 ...... 24 ix

Figure 1.17 Aminoketyl radicals and c and z• ions by ECD2, 84 ...... 24

Figure 1.18 Mass spectrum of ubiquitin ([M+9H]9+) by ECD FTICR with 9.4 T instrument.89 ...... 25 Figure 1.19 Schematic of a single neutral injection line (a)42 and a multiported pulsed valve interface (b)90 of a linear quadrupole ion trap for ion/molecule reactions...... 27 Figure 1.20 Schematic of the differentially pumped dual LQIT with ion optics and differential pumping. LQIT1 and LQIT2 are the ﬁrst and second linear quadrupole ion traps (LQIT).91 ...... 28

Figure ...... Page Figure 1.21 Schematic of a multi-source quadrupole ion trap instrument containing two electrospray ionization (ESI) sources and one atmospheric sampling glow discharge ionization (ASGDI) source interfaced.98b ...... 30 Figure 1.22 Schematic of a triple quadrupole/linear ion trap modified by addition of an atmospheric sampling glow discharge ionization source on the side of Q3 linear ion trap for ion/ion reactions.100 ...... 31 Figure 1.23 Schematic of (a) a 3D ion trap coupled with a fast atom bombardment (FAB) gun for metastable atom-activation dissociation19 and (b) a laser coupled linear ion trap for infrared multiple photon dissociation (IRMPD)102b and ultraviolet photon dissociation (UVPD)103...... 32 Figure 1.24 Schematic of a modified quadrupole time-of-flight tandem mass spectrometer for ion/ion reactions with dual ion source.104 ...... 34 Figure 1.25 Schematic of an Orbitrap Elite.107a ...... 35 Figure 1.26 Schematic of (a) conventional linear ion trap mass spectrometer with differential pumping system and ion optics and (b) miniature DAPI-RIT mass spectrometers highlighted Mini 11 mass spectrometer of 9 pounds and Mini 12 of Point-of-care personal mass spectrometer.110, 113 ...... 36 Figure 1.27 (a) Scan function for mass analysis using a DAPI mass spectrometer. (b)

Manifold pressure measured during scanning, with an open time of 20 ms and x

a closed time of 850 ms for DAPI.110 ...... 37 Figure 1.28 Schematic of a DAPI-RIT-DAPI mass spectrometer coupled with a pyrolysis radical source for the study of gas-phase ion/radical reactions...... 39 Figure 2.1 (a) Schematic representation of the DAPI-RIT-DAPI system; (b) General waveforms of the in-trap gas-phase reactions ...... 55 Figure 2.2 Ion/molecule reactions: thiyl radical cations react with allyl iodide ...... 56

Figure ...... Page Figure 2.3 (a) Ion/ion reactions: Angiotensin I triply charged cations react with deprotonated pentachlorophenol anions (insert: Isolation of the triply charged species); (b) MS spectrum of Angiotensin I without opening DAPI-2; (c) MS spectrum of isolated triply charged peptide ions with DAPI-2 opening but the nanoESI source being turned off for anion production; and (d) aveforms of the ion/ion reaction (dc was set to -10 V during mass analysis)...... 58 Figure 2.4 CID spectra and MS/MS efficiency of protonated cocaine m/z 304 at different pressures. Activation time 30 ms, amplitude 200 mV...... 60 Figure 2.5 Ion-trap CID spectra of deprotonated 9-anthracenecarboxylic acid at (a) 6 mTorr 200 ms delay, (b) 2 mTorr 500 ms delay, (c) 1 mTorr 780 ms delay, from the DAPI opening on DAPI-RIT-DAPI system. The yellow, red, and blue columns illustrate the positions of ion introduction, dipolar ac excitation, and MS scan, respectively; (d) Ion-trap CID spectrum of m/z 195 on DAPI- RIT-DAPI system; (e) Ion-trap CID spectrum of deprotonated 9- anthracenecarboxylic acid on QTRAP 4000; (f) Ion/molecule reactions of deprotonated anthracene ions with water vapor on QTRAP 4000. The CID activation time was set to 30 ms on DAPI-RIT-DAPI system...... 62 Figure 2.6 Energy characterization of DAPI. (a) Calculated survival ion yields and (b)

Internal energy distributions of DAPI vs. continuous API for ion introduction; xi

Fragmentation spectra of thermometer ions after opening DAPI-2 for 20 ms with capillary 3 (c) i.d. 0.5 mm and (d) i.d. 0.75 mm. *p-R represents the

fragment of p-R (R = OCH3, CH3, Cl, CN, NO2)...... 64 Figure 2.7 Ion/molecule reactions of peptide thiyl radical ions and dimethyl disulfide. (a) MS spectrum of S-nitrosoglutathione; (b) Thiyl radical (m/z 307) produced by CID of protonated S-nitrosoglutathione; (c) Reactions between thiyl radicals and dimethyl disulfide (Capillary 3 i.d. 0.125 mm; DAPI-2 opening time 25 ms; q=0.35 for thiyl radical ions); (d) Ion/molecule reactions between isolated m/z 307 from panel (c) with dimethyl disulfide...... 66

xii

Figure ...... Page Figure 2.8 Thiyl radical reaction for different capillary inner diameters and opening times...... 67 Figure 2.9 MS spectra of thiyl radical reaction for activation with ac amplitude (a) 0.06 V; (b) 0.066 V; (c) 0.07 V; and (d) CID spectrum of thiyl radical ions...... 68 Figure 3.1 (a) Schematic of the home-built platform for the study of reactive radical species and (b) the general MS scan function for ion/radical reactions (y-axis: voltage). DAPI: discontinuous atmospheric pres-sure interface; RIT: rectilinear ion trap; GDEI: glow discharge electron impact; rf: radio frequency; ac: alternative current...... 75 Figure 3.2 Carbene reaction spectra: (a) MS2 reaction of protonated n-heptylamine with heated 1,3-dibromopropyne; (b) MS3 CID of the reaction product; (c) MS2 2 reaction of protonated n-heptylamine with heated d12-pentane; and (d) MS reaction of protonated n-heptylamine 18C6 complex with heated 1,3- dibromopropyne...... 79 Figure 3.3 MS2 reaction spectra of heptyl ammonium and heated (a) allylchloride, (b) propargylbromide, (c) 1,5-hexadiene, and (d) pentane...... 80 Figure 3.4 MS2 reaction spectra of protonated (a) dibutylamine, (b) tributylamine, and (c) cetyltrimethyl ammonium with heated propargylbromide, and MS3 CID spectra of the reaction products for (d) dibutylamine and (e) tributylamine. 81 xii

Figure 3.5 MS2 reaction spectra of (a) protonated and (b) sodiated proline methyl ester with heated allylchloride, and their corresponding product MS3 CID spectra (c) and (d), respectively...... 82 + Figure 3.6 Calculated PES of (a) CH3NH3 /c-C3H2 reaction to form a C-N covalent bond from Brauman double-well complexes (numbers: CCSD(T)/aug-cc-pVTZ, kJ/mol) and (b) the two Brauman intermediates as a function of the distance of N-H---C, blue square: CCSD(T)/aug-cc-pVTZ single point energies on M06-2X/6-311+G(2d,p) optimized geometries; open circle: M06-2X/6- 311+G(2d,p) optimized; filled circle: B3LYP/6-311+G(2d,p) optimized. .... 85 Figure 3.7 Calculated PES of N--H--C angle graph of Complex 1, 2 and product A ...... 86

xiii

Figure ...... Page Figure 3.8 The breakdown curve of protonated butylamine/carbene reaction by collisional activation: relative intensity of parent complex ([C], blue circle), covalent product ([C-40], red diamond), proton-transfer product (m/z 39, green triangle), and direct dissociation ([C-38], green square) as a function of activation energy...... 87 Figure 3.9 Selected MS3 reaction product CID spectra of (a) butylamine, (b)

ethylmethylamine, (c) dipropylamine, and (d) guanidine with c-C3H2 and

(e) %Covalent of protonated alkyl amines/c-C3H2 reactions as a function of

GBs of the corresponding alkyl amines. The %Covalent is calculated as I[c-

40]/(I[39]+I[c-40]+I[c-38]), where I[39], I[c-40], and I[c-38] correspond to the intensity of m/z 39 as the proton-transfer product, neutral loss of 40 Da as the covalent product, and neutral loss of 38 Da as the direct-dissociation product, respectively, in the MS3 CID spectra based on 80-90% parent ions fragmented. Error bars were based on 5 duplicates of an average of 3 spectra...... 90 3 + Figure 3.10 MS reaction product CID spectrum of C3H3 and neutral n-heptyl amine ... 91 Figure 4.1 MS2 carbene reaction spectra of (a) protonated adenine and (c) protonated adenosine; and MS3 CID spectra (b) adenine carbene monoadduct and (d)

xiii adenosine carbene monoadduct ...... 113

Figure 4.2 MS2 carbene reaction spectra of (a) protonated uracil and (b) protonated uridine; and MS3 CID spectrum (c) uridine carbene monoadduct ...... 114 Figure 4.3 (a) MS2 carbene reaction spectrum of protonated Lys, (b) MS3 CID spectrum of Lys/carbene reaction product, and (c) proposed fragmentation pathway of the reaction product...... 119 Figure 4.4 MS2 carbene reaction spectra of (a) protonated Arg and (c) singly charged MRFA; MS3 CID spectra of (b) Arg/carbene reaction product and (d) MRFA/carbene reaction product...... 120 Figure 4.5 MS2 reaction spectra of triply charged Angiotensin I with heated allylchoride at (a) 750, (b) 800, and (c) 900 degree Celsius...... 122

xiv

Figure ...... Page Figure 4.6 (a) MS1 spectrum of multiply-charged Ubiquitin, and (b) MS2 spectrum of its reaction with heated allylchoride...... 123 Figure 4.7 MS2 carbene reaction spectra of (a) protonated acetyl cysteine, (b) protonated cystine, and (c) singly charged oxidized glutathione...... 124 Figure 4.8 MS2 reaction spectra of (a) hydroxyl group containing choline and (b) double bond containing LPC (18:2) sodium adduct. No reaction product was observed...... 125

xiv

ABBREVIATIONS

ac alternative-current

APCI atmospheric pressure chemical ionization

API atmospheric pressure interface

ASGDI atmospheric sampling glow discharge ionization

B magnetic field

BIRD blackbody infrared dissociation c-C3H2 cyclopropenylidene

C3H2 vinylidene carbene

CAD collision-activated dissociation

CI chemical ionization

CID collision-induced dissociation xv

DAPI discontinuous atmospheric pressure interface dc direct-current

E electric field

ECD electron capture dissociation

EDD electron detachment dissociation

EI electron impact

ESI electrospray ionization

ETD electron transfer dissociation

xvi

FA flowing-afterglow

FAB fast atom bombardment

FT-ICR Fourier transfer ion-cyclotron-resonance

GB gas-phase basicity

GDEI glow discharge electron impact

GIB guided ion beam

H/D hydrogen/deuterium exchange

IR infrared

IRMPD infrared multiphoton dissociation

ISM interstellar medium

LIAD laser-induced acoustic desorption

LIT linear ion trap

LMCO low mass cut-off

LQIT linear quadrupole ion trap

LTP low temperature plasma xvi

MAD metastable atom-activated dissociation

MALDI matrix-assisted laser desorption ionization

MIKES mass-analyzed ion-kinetic-energy spectrometry

MRM multiple reaction monitoring

MS mass spectrometry

MSn tandem mass spectrometry

OH hydroxyl radical

PD photon dissociation

xvii ppm parts per million pptv parts per trillion volume

PTM posttranslational modification

QqQ triple-quadrupole

QqQqQ penta-quadrupole rf radio frequency

RIT rectilinear ion trap

ROS reactive oxygen species

SIFT selected-ion flowing-tube

SORI sustained off-resonance irradiation

SRM selected reaction monitoring

TOF time-of-flight

UV ultraviolet

UVPD ultraviolet photon dissociation

VOC volatile organic compound xvii

xviii

ABSTRACT

Lin, Ziqing. Ph.D., Purdue University, May 2015. Instrumentation and Development of a Mass Spectrometry System for the Study of Gas-Phase Biomolecular Ion Reactions. Major Professor: Zheng Ouyang.

Gas-phase reactions of biomolecular ions are highly relevant to the understanding of structures and functions of the biomolecules. Mass spectrometry is a powerful tool in investigating gas-phase ion chemistry. Various mass spectrometers have been developed to explore ion/molecule reactions, ion/ion reactions, ion/photon reactions, ion/radical reactions etc., both at atmospheric pressure and in vacuum. In-vacuum reactions have an advantage of involving pre-selecting the ions for the reactions using a mass analyzer.

Over the decades, a variety of mass analyzers have been employed in the research of ion

chemistry. Hybrid configurations, such as quadrupole ion trap with a time-of-flight and or xviii a quadrupole ion trap tandem with an Orbitrap, have been utilized to improve the performances for both the reaction (in trapping mode) and the mass analysis (accurate mass measurements).

Complicated instrument structures, including ion optics, multiple mass analyzers and differential pumping for high vacuum, are typically required for the mass spectrometers for gas phase ion chemistry study. An alternative approach is to simplify the instrumentation by using pulsed discontinuous atmospheric pressure interfaces for introducing ionic or neutral reactants and a single ion trap as both the reactor and the

xix mass analyzer. Such a simple mass spectrometry system was set up and demonstrated using two discontinuous atmospheric pressure interfaces in the study for this thesis. It was capable of carrying out ion/molecule and ion/ion reactions at an elevated pressure without the needs of ion optics or differential pumping system. Together with a pyrolysis radical source, in-vacuum ion/radical reactions were performed and their associated chemistry was studied. Radicals are important intermediates related to biochemical processes and biological functions. There are very limited techniques to monitor the reactive intermediates in-situ during a multi-step reaction in aqueous phase. On the other hand, these intermediates can be “cooled down” and preserved into a single-step procedure in gas-phase reactions since they only occur via collisions. Therefore, the fundamental study of gas-phase radical ion chemistry will provide evidences of the reactivity, energetics, and structural information of biological radicals, which has the potential to solve puzzles of aging, disease biomarker identification, and enzymatic activities.

Using the system described above, a new reaction between protonated alkyl amines xix

and pyrolysis formed cyclopropenylidene carbene was discovered, as the first experimental evidence of the reactivity of cyclopropenylidene. Given the important role of cyclopropenylidene in the combustion chemistry, organic synthesis, and interstellar chemistry, it is highly desirable to establish a fundamental understanding of their physical and chemical properties. The amine/cyclopropenylidene reactions were systematically studied using both theoretical calculation and experimental evidences. A proton-bound dimer reaction mechanism was proposed, with the amine and the carbene sharing a proton to form a complex as the first step, which was closely related to the high gas-

xx phase basicity of cyclopropenylidene. Subsequent unimolecular dissociation of the complex yielded three possible reaction pathways, including proton-transfer to the carbene, covalent product formation, and direct separation. These reactions were studied with a variety of alkyl amines of different gas-phase basicities. For the covalent complex formation, partial protonation on cyclopropenylidene within the dimer facilitates subsequent nucleophilic attack to the carbene carbon by the amine nitrogen and leads to a

C-N bond formation. The highest yield of covalent complex was achieved with the gas- phase basicity of the amine slightly lower but comparable to cyclopropenylidene. The results demonstrated a new reaction pathway of cyclopropenylidene besides the formation of cyclopropenium, which has long been considered as a “dead end” in interstellar carbon chemistry.

Further reactivity study of cyclopropenylidene towards biomolecular ions was also carried out for nucleobases, nucleosides, amino acids, peptides, proteins, and lipids. The reaction to form proton-bound dimer for protonated biomolecular ions remained as the

dominant reaction pathway. Interestingly, other possible reaction pathways, such as xx

modifications of thiyl group or disulfide bonds, double bond addition, and single bond insertion, were inhibited in gas-phase ion/carbene reactions. Such results inferred that the reactivity of neutral species was not directly applicable to ion reactions, with the proton involved in the gas-phase biomolecular ion reactions.

CHAPTER 1. INTRODUCTION

1.1 Gas-phase biomolecular ion reactions and mass spectrometry instrumentations

Gas-phase ion reactions involve interactions between ions and various species including molecules,1 electrons,2 ions,3 photons,4 reactive organic species,5 etc. Mass spectrometry (MS), which analyzes charged particles by their mass-to-charge ratio (m/z), is naturally involved with gas-phase ion chemistry and has been a powerful tool for manipulating and analyzing gas-phase ions. Back in the 1950s’, researchers made great efforts to decrease the pressure inside the vacuum chamber of mass spectrometers to improve the resolution, accuracy and sensitivity. In the 1970s’, R. Graham Cooks at

Purdue University proposed and developed the early generations of tandem MS instruments for complex mixture analysis using in-vacuum gas collisions.6 The most

common tandem MS technique is known as collision-induced dissociation (CID, a.k.a. 1

collision-activated dissociation, CAD), which utilizes collisions to break the selected ions into fragments.7 These characteristic fragments, acting as fingerprints of the parent ion, provide unambiguous structural identification of target analytes. The development of CID promoted MS as a “gold standard” in chemical analysis in terms of both qualification and quantification. The nature of CID is an energy-transfer ion/molecule reaction: the kinetic energy of the ions accelerated by the electric/magnetic field through collisions between the ions of interest and the inert buffer gas molecules, converts into internal energy, further cleaving the weakest linkages of the ions to generate the fragments. The study of

2 gas-phase reactions has been growing for both studies of basic chemistry and application development in the past two decades. The advantage of gas-phase reactions, compared to those in aqueous or liquid phase, is that the reaction occurs via direct collisions without solvent clusters surrounded, where the energy barrier is lower, making it both kinetically and thermodynamically favorable, e.g. proton-transfer reaction occurs more readily in gas phase than in aqueous phase (Figure 1.1).8

Figure 1.1 Proton-transfer reaction in the gas-phase vs. the aqueous phase

Owing to the two revolutionary ionization techniques: electrospray ionization (ESI)9 and matrix assisted laser desorption/ionization (MALDI)10, which have been awarded the

Nobel Prize in Chemistry in 200211, intact ions of biomolecules such as peptides and proteins can be produced from their condensed phase directly to the gas phase. MS was then adopted for biomolecular identification and became the method of choice in proteomics to study the structures and functions of proteins.12 The primary structure of a peptide could be sequenced through the tandem MS methods.13 Two decades later, a variety of dissociation methods have thrived in proteomics using different types of gas-

3 phase reactions.13 Table 1.1 summarizes the typical dissociation techniques, their corresponding gas-phase reaction types, and the mass spectrometers that were involved in protein sequencing. It demonstrates how gas-phase ion reactions, together with the development of MS instrumentations, are closely linked to identifying biomolecules and searching for disease biomarkers.6 Today, almost all of the proteomic strategies are based on MS because it allows high throughput and structural analysis with high sensitivity.13-14

Table 1.1 Tandem mass spectrometry dissociation techniques in proteomics Tandem MS Gas-phase reaction type Instruments Triple Collision induced Ion/molecule quadrupoles, dissociation (CID) [M + nH]n+ + A → fragments ion traps15 Fourier transfer Ion/electron Electron capture ion-cyclotron- [M + nH]n+ + e- → [M + H• + (n-1)H](n-1)+ dissociation (ECD) resonance (FTI- → fragments CR)16 Ion/ion Electron transfer [M + nH]n+ + A•- → [M + H• + (n-1)H](n-1)+ Ion traps17 dissociation (ETD) + A → fragments

Ion/photon Proton dissociation [M + nH]n+ + hv → [(M + nH)n+]*→ Ion traps18 (PD) fragments

Metastable atom- Ion/metastable species activated dissociation [M + nH]n+ + A* → [(M + nH)n+]* + A → Ion traps19 (MAD) fragments Fourier transfer Electron-detachment Ion/electron ion cyclotron dissociation [M - nH]n- + e- → [M -H• - (n-1)H](n+1)- → resonance (FT- (EDD) fragments ICR) 20 *Precursor peptide/protein ions shown in bold

Among all the gas-phase interactions in bioanalysis, reactions involving radicals have been increasingly drawing attentions.5 Radical ions, which consist of at least one unpaired electron are significantly more reactive than even-electron species, would

4 induce more gas-phase chemistry with two reactive functional groups within one species: the charge and the radical sites. In living organisms, radicals are important intermediates that are highly relevant to biochemical processes and biological functions. For instance, radicals initiate modifications in proteins,21 DNAs,22 and lipids23 at the molecular level, which are believed to be the cause of cell damage,24 cell death,25 aging,21a, 26 neurodegenerative diseases,25-26 and cancers27. Besides the damaging effects, radicals also contribute to the catalytic activities of enzymes in several classes.28 Taking ribonucleotide reductase as an example, a thiyl radical was used for catalysis,28b however, the catalytic center is more than ten amino acid residues away from the radical site.28c

The puzzle of how the radical activity is controlled by the enzyme via such a long- distance radical transfer process remains unsolved at the current stage. This is due to the very limited techniques to monitor the reactive intermediates in-situ during a multi-step reaction in the aqueous phase. On the other hand, these intermediates can be “cooled down” and preserved into a single-step procedure since gas-phase reactions only occur via collisions. Therefore, the fundamental study of gas-phase bio-radical reactions would

provide direct and/or indirect evidences of the reactivity, energetics, and structural information of biological intermediates. The study might explain the intra- and intermolecular radical transfer in biological systems, and could potentially help to develop drugs to cure serious diseases. Thus, developing a MS system suitable for gas-phase reactions would be the very first step in achieving these goals. This review will start with the concept of tandem mass spectrometry (MSn) and introduce gas-phase reactions at different pressure stages of a tandem mass spectrometer: (1) the studies of gas-phase bio-

5 radical chemistry at atmospheric pressure and (2) MS instrumentations for gas-phase reactions in vacuum.

1.1.1 Tandem mass spectrometry in gas-phase ion reactions

Tandem mass spectrometry (MSn) is a stepwise mass spectrometry analysis.7a A direct MS analysis of ions produced from the ion source according to their m/z values is called an MS full scan, also known as MS1. Selected precursor ions can undergo reactions or fragmentations between two MS analysis stages, and the resulting product ions can be analyzed in the next MS stage to generate MS2 spectra. Using a variety of mass analyzers,

MSn has been achieved with i) tandem-in-space mode, using sectors,29 triple quadrupoles,30 and time-of-fight (TOF)31; or ii) tandem-in-time, using quadrupole ion traps32 and Fourier transform ion cyclotron resonance (FT-ICR)33. Figure 1.2 gives a brief summary of these MSn techniques.

Figure 1.2 Tandem mass spectrometry in space vs. in time

For the study of bio-radical (biomolecules that contain at least one unpaired electron) chemistry, one additional reaction step must be applied to the biomolecules to create a radical ion since soft ionization methods such as electrospray (ESI) and nanoelectrospray (nanoESI)34 usually generate lower-energy, even-electron species rather than odd-electron radical ions. Bio-radical ions can be made directly at atmospheric pressure through the interactions between the whole ion population and free radicals or alternatively in vacuum through further reactions of the selected ionic species. Product ions of ion/radical reactions at atmospheric pressure, for example in ESI plume, could simply be introduced to the mass analyzer through atmospheric pressure interface (API) and focusing lens. As a result, only MS1 is required to obtain a reaction spectrum.

Tandem mass spectrometry might be further applied to elucidate the molecular structure of the product ions, as employed in almost all of the commercial MS systems. It normally does not need modifications of the MS instruments; however, side reactions can occur with multiple ionic reactants. Therefore, gas-phase ion reactions in vacuum with the pre- selection of ions are preferred. Isolation of the precursor ions is performed before the

reaction and the reaction spectra are acquired in MS2. Yet, introducing a second flow of neutral radicals or another ionic species in vacuum for reactions could be challenging and usually requires extensive instrumentational modification. In the next two sections, strategies for performing gas-phase ion reactions at atmospheric pressure and in vacuum will be discussed.

1.1.2 Gas-phase biomolecular ion/radical reactions at atmospheric pressure

The interaction of biomolecular ions and free radicals can be studied in the nanoESI plume or in the spray bulk solution in atmosphere by adding a radical source such as a low temperature plasma (LTP) generator35 (Figure 1.3a) and an ultraviolet (UV) lamp36

(Figure 1.3b) aside the ion source. The plasma or UV irradiation generates reactive organic species (ROS) e.g. hydroxyl radicals (OHs), which subsequently react with the ions and charged droplets in the nanoESI plume, or directly in the bulk solution. The high number density and large cross-section at atmospheric pressure (760 Torr) facilitate the reactions between two species. Rich chemistry can be observed through this simple reaction approach for biomolecules such as disulfide-linked peptides and unsaturated lipids. Note that although there is no restriction on MS instrumentation for these reactions taking place before the product ions are introduced to the mass analyzer, MS systems with versatile dissociation methods, e.g. beam-type CID or ion-trap slow-heating CID,37 could be essential to study the structure, reactivity, and further applications of the reaction products.

Figure 1.3 Radical reactions in the nanoESI plume and bulk solution using low temperature plasma (LTP)35 or ultraviolet (UV) lamp36.

1.1.2.1 Peptides

Disulfide linkage (S-S) is a type of posttranslational modification (PTM) formed by the oxidation reaction between two cysteine residues among intra- or inter- peptide chains.

It is responsible for the stabilization of the native structures of proteins.38 The disulfide bonds are considered difficult to break using CID due to its bonding energy being significantly higher than those of the peptide backbones. Therefore, intra-molecular disulfide bonds increase the difficulty in sequencing using even-electron based dissociation methods.39 The conventional method for analyzing disulfide peptides is to cleave the disulfide bonds using wet chemistry before sequencing (reducing disulfide bond by alkylation39a). However, this causes the loss of constructional information.

Alternative methods are to use odd-electron radical-based dissociation methods (CID,40

ECD,16 ETD,17 EDD,20a, 20b UVPD,41 MAD,19 and etc.) in vacuum through additional gas- phase reactions. Fortunately, disulfide bonds are very sensitive to free OHs, producing different types of radical products (such as carbon-centered versus heteroatom-centered radicals) with highly specific radical sites. As an example (Figure 1.4), the cleavage of a

disulfide bond occurs with the attack by hydroxyl radicals which yields three sets of products: thiyl (-S•), sulfinyl (-SO•), and perthiyl (-SS•) radical ions.42

Figure 1.4 Reactions of protonated disulfide peptide and oxygen-centered radicals5 to form thiyl (-S•), sulfinyl (-SO•), and perthiyl (-SS•) radical ions in nanoESI plume: (a) peptide chain CLPTR of HMAC-CLPTR, and (b) chain AGCK of AGC(TFTSC)K.42

Among the three radical species generated by the reaction in the nanoESI plume, thiyl radicals have the highest reactivity while sulfinyl radicals are the most inert.42 Although the cysteine sulfinyl radicals have low reactivity in aqueous solvents or under gas-phase

bimolecular reaction conditions, additional energy could trigger intramolecular reactions.

The energy applied by collisional activation can be sufficient to conquer the reaction energy barrier but barely enough to cause backbone cleavage. Two reaction pathways of radical migration were observed for a cysteine sulfinyl radical (SO•Cys) with either a free thiol (-SH)43 or a disulfide bond (S-S)44 inside one peptide sulfinyl ion (Figure 1.5). The discovered reaction channels indicate: (1) the disulfide bond scrambling could happen in a protein based on the radical migration; (2) protein conformation and structural change would be possible by the attack of the cysteine sulfinyl radicals; and (3) an oxidative

10 damage in terms of forming sulfinyl radicals, might potentially be cured with a nearby free thiol group43.

Figure 1.5 Intramolecular cysteine sulfinyl radical (SO•Cys) transfer with (a) a free thiol43 and (b) a disulfide bond44

Besides the reactivity study of bio-radical ions, ion/radical reactions in the ESI plume enhance the backbone cleavage, thus improving the sequencing coverage.35 The backbone cleavage for insulin, a protein with three disulfide bonds, occurred at an 10

efficiency rate of 26% obtained from even-electron based CID, but improved to about 60% with odd-electron induced ECD or ETD methods.45 A favorable value of 75% backbone cleavage has been achieved for cleaving disulfide bonds followed by applying a conventional CID after the radical reaction in the ESI plume (Figure 1.6).35

Figure 1.6 Coupling radical reactions in the ESI plume to cleave the disulfide bonds, with a subsequent MS2 scan to improve sequence information of disulfide peptides5, 35

1.1.2.2 Lipids

Lipids are a group of biomolecules that function as the building blocks of cell membranes, energy storage, and signaling transduction in living organisms.46 Individual lipid47 or the lipid profile48 has been used as biomarkers for cancer diagnosis by ambient ionization MS techniques, assuming that the lipid bio-synthesis pathways have been 11

changed in the tumor tissues.46 The degree of unsaturation in lipids (the number of C=C bonds) and the location of double bonds determine the biomolecular structure and the biological functions.49 Even-electron based dissociation methods are very helpful in identifying the head group, the acyl chain composition, and the number of C=C bonds.50

However, the positions of C=C bonds, on the other hand, are difficult to be determined since the bonding energy is much higher for C=C double bonds (~keV) than C-C single bonds. This problem could not be solved with high energy collisions by sector or TOF-

TOF instruments because the C-C bonds are cleaved before the C=C bonds in a lipid. In order to differentiate the unsaturated lipid isomers (different double positions in the acyl chain), radical chemistry has been applied in vacuum or at atmospheric pressure. Radical- directed dissociation (RDD) of lipid ions in vacuum yields fragmentation patterns revealing information of double bond positions: a photo-caged radical precursor releases the radical by irradiation, which forms a complex with the unsaturated lipids and fragments are subsequently generated through CID.51 Alternatively, a Paternò–Büchi (PB) reaction can be performed in the bulk solution of the nanoESI spray tip by UV lamp irradiation (Figure 1.3b).52 A four-member ring is first formed through the interaction of a ketone/aldehyde with an olefin, thus creating a set of low energy covalent bonds which are then cleaved in the retro P-B reaction as shown in Figure 1.7.

Figure 1.7 Scheme of Paternò–Büchi (PB) reaction between ketone/aldehyde and olefin together with possible retro P-B reactions.52

In the analysis of oleic acid (Figure 1.8),52 a covalent adduct m/z 339 of P-B reaction of deprotonated oleic acid and acetone was formed with the 254 nm UV irradiation

(Figure 1.8a, b). MS2 CID of the product clearly showed three peaks in Figure 1.8c: m/z

281 corresponding to the retro P-B reaction to produce the original reactants

(deprotonated oleic acid); and one set of diagnostic ions with a mass difference of 26 Da

13 resulting from the fragments (structures 3 and 5) of two possible arrangements of P-B reaction (isomers 1 and 2) (Figure 1.8d). By this means, lipid isomers (fatty acids and phospholipids) in tissue samples with different double bond locations have been identified and potential lipid biomarkers of diseases are under investigation.

Figure 1.8 On-line coupling of P-B reactions of oleic acid and acetone with negative MS. 1 2

MS spectra of oleic acid (a) before P-B reaction (b) after P-B reaction; MS CID of the 13

P-B reaction products at (c) m/z 339.3; and (d) structures of the diagnostics ions (structures 3 and 5). The structures explain the origin of the 26-Da mass difference.52

1.1.3 Instrumentations for gas-phase ion reactions in vacuum

Compared to the reactions occurring at atmospheric pressure, gas-phase ion reactions can also be carried out in vacuum. It provides one huge advantage of controlling variables by having a single ionic reactant selected from the ion population, which largely decreases the uncertainty of side reactions by unknown ions generated by the ion source.

The tradeoffs are however, the necessity of instrumentational modification and the relatively low number density in vacuum (enough for reactions to occur) due to the low pressures (usually 10-3 to 10-7 Torr). The first stage of tandem mass spectrometry (MS1) can be used to isolate the precursor ions based on their m/z values. The subsequent gas- phase reactions are then carried out in the following MS stages (MSn). Several types of mass spectrometers have been modified to study gas-phase ion chemistry. For tandem-in- space mass spectrometers such as triple-quadrupoles, penta-quadrupole, and selected-ion flow-tube, a reaction chamber (designed for collisions) and at least one additional mass analyzer for the following product analysis are required. On the other hand, the reactor and mass analyzer can be integrated for trap-type instruments such as ion traps and

Fourier transform ion cyclotron resonance, which are able to store ions for a relatively long reaction time and allow subsequent tandem MS. Hybrid instruments, involving a trapping mode reaction chamber followed by a high accuracy mass analyzer for mass analysis, are suitable for ion manipulation and reactions in the vessel, then providing a

high resolution product scan. Nevertheless, not all of these research prototype 14

instruments have been applied in gas-phase biomolecular ions reactions, especially for bio-ion/radical reactions. This section will have an emphasis on the instrumentation: the strategies to bring two species inside vacuum and the related reactions not limited to biological ion interactions.

1.1.3.1 Sectors and time-of-flights (TOFs)

A sector mass spectrometer is a MS system using a static electric (E), a magnetic (B) field, or a set of the combination of the two sectors to separate ions as a mass analyzer. In

15 the case of mass-analyzed ion-kinetic-energy spectrometry (MIKES),6a a magnetic sector followed by an electronic sector was originally designed to study kinetic energy release associated with metastable ion fragmentation by measuring the kinetic energy of mass- selected ions. This instrument is most famously recognized as the first generation of tandem mass spectrometer,6b, 53 with collision gas introduced between the two sectors

(Figure 1.9). The collisional energy in MIKES tandem MS could be as high as ca. keV, which is sufficient to obtain fragments from most ionic species during tandem MS.

Guided ion beam (GIB) tandem MS is another sector instrument involved in gas-phase chemistry. The ion beams selected by a magnetic sector are introduced to the reaction cell

(octapole ion guide) with reactant neutral of choice, followed by a double focusing sector54 or a quadrupole55 as mass analyzer. This instrument has been widely used for the determination of thermodynamic information for bimolecular reactions and CID.55

Figure 1.9 Schematic of mass-analyzed ion-kinetic-energy spectrometry (MIKES) with collision gas for chemical mixture analysis6a

As for a tandem time of flight56 (TOF-TOF, shown in Figure 1.10), the collisional energy is able to reach keV level, similar to that of the sectors. Although TOF-TOF

16 instruments have been barely used in research of gas-phase ion reactions, TOF-TOF provides a good dissociation methods coupled with MALDI for peptide identification.57

Furthermore, high mass resolution capability of TOF analyzer serves as an excellent product mass analyzer for in-vacuum gas-phase reaction in hybrid instruments, which will be further discussed in section 0.

Figure 1.10 Schematic of tandem time of flight (TOF-TOF) mass spectrometer56

1.1.3.2 Tandem quadrupoles

The triple-quadrupole (QqQ) mass spectrometer was developed in late 1970s30 and remains as an outstanding MS2 instrument for chemical quantification. Triple- quadrupoles have demonstrated tremendous capabilities in performing a set of MS2 experiments in tandem-in-space mode in three steps: (1) isolating precursor ions in Q1 by applying certain amplitudes of radio-frequency (rf) and direct-current (dc) to allow ion transmission at a narrow m/z window; (2) dissociative or reactive collisions in rf-only collisional quadrupole q2 with activation energy ranging from 0 to tens of eV when using

17 inert or reactive buffer gas; and (3) product ion scan in Q3. The use of triple-quadrupoles in studying ion/molecule reactions is valuable since the information of both precursors and products are provided through different MS2 scan modes such as product ion scan, precursor ion scan, neutral loss, and selected reaction monitoring (SRM) or multiple reaction monitoring (MRM).58

Figure 1.11 Schematic of penta-quadrupole (QqQqQ) mass spectrometer59

The limitation inherent to triple-quadrupoles for ion/molecule reaction study is the lack of structural analysis of the product ions with only two MS stages. Therefore, a simple way to improve tandem-in-space instruments for ion/molecule reactions is to add

17 3 60 another MS stage, MS . The penta-quadrupole (QqQqQ) (Figure 1.11) was designed specifically for MS3 analysis for ion/molecule reactions. It employs two reaction rf-only quadrupoles (q2 and q4) for reactions, and three rf/dc quadrupoles (Q1, Q3, and Q5) for

MS analysis. The tandem-in-space arrangement allows the study of ion/molecule reactions involving mass-selected ions in two separate reaction regions (q2 and q4) by using Q1, Q3, and Q5 for mass analysis. The first reaction region q2 can be set as dissociative-collisional chamber for CID whereas q4 was filled with neutral reactants for reactive collisions, or vice versa. A total of 15 different types of MS3 scan functions61 can

18 be achieved with 0-3 dimensions as shown in Figure 1.12. Each circle represents the operation of one of the three mass analyzers, with a filled circle for a mass selection and an open circle for MS scanning. Note that with all three mass analyzers transmitting fixed masses, the consecutive reaction monitoring (three filled circles, 0 dimension) provides mass spectra of one dimension. A remarkable 4D mass spectra were therefore acquired using the entire MS3 data domain.62

Figure 1.12 Symbols and names given to QqQqQ MS experiments with 0, 1, 2, and 3 dimensions. A ﬁxed mass (ﬁlled circle) or variable masses (open circle) can be chosen for reactions.61

An example of 3D familial scan is demonstrated in Figure 1.13.63 The ions produced from 3-octanone by electron impact (EI) underwent CID in q2 via collisions with Argon

19 gas and the CID products of m/z 43 were selected for reacting with isoprene in q4; finally the ion/molecule reaction product ions were analyzed by Q5. This experiment was useful for ion chemistry since it provided reactivity information of isobaric and isomeric ionic populations. Both sequential product spectra (horizontal) and sequential precursor spectra

(angles) can be extracted. Figure 1.13b shows the sequential precursors generating m/z 43 reacting predominantly by proton transfer that led to the product ions of m/z 81, whereas

Figure 1.13c identifies the sequential precursors producing acetyl cations of m/z 43, which reacted with isoprene to form [4+2+] cycloadduct of m/z 111.

Figure 1.13 (a) Penta-quadrupole three-dimensional triple-stage (MS3) familial spectrum for 3-octanone ions acquired by selecting ions of m/z 43 as the intermediates. The angled Q1 axis displays the precursors of m/z 43 ions, whereas the horizontal Q5 axis displays the ion/molecule products of these CID-generated m/z 43 ions arising from reactions with isoprene. Triple-stage sequential precursor spectra extracted from the 3D familial spectrum by selection in Q5 of the ﬁnal products of (b) m/z 81 and (c) m/z 111.63

Penta-quadrupole MS system has been proven to be suitable to study various ion/molecule reactions.60 However, other types of gas-phase reactions such as ion/ion and ion/radical reactions could barely be done on the penta-quadrupole mass spectrometer without further modifications.

1.1.3.3 Flowing-afterglow selected-ion flow-tube (FA-SIFT)

Selected-ion flow-tube mass spectrometer has been used to study ion/molecule reactions64 and also for quantitative analysis of trace, volatile organic compounds (VOCs) using chemical ionization (typically proton-transfer reactions) with the selected precursor ionic species along a drift tube within a well-controlled period. The applications extend to gas analysis,65 breath testing,66 VOC monitoring from natural products67 etc., with pptv level of detection limit. Together with the flowing-afterglow (FA) techniques, selected- ion flow-tube has been employed to measure numerous ion-molecule kinetics data especially those related to interstellar chemistry at a low temperature.68 In an FA-SIFT

instrument, the precursor ions generated by EI ionizer in the source flow tube (FA) are sequentially selected by a SIFT quadrupole mass filter. The mass-selected ions flow through the reaction tube where ion/molecule reactions occur with the neutral reactant being introduced into the flow tube. The resulting product ions are scanned in the detection quadrupole mass filter to produce the MS2 spectra. With the well-defined flow time inside the reaction tube, the ion/molecule reaction kinetics can be acquired by introducing neutrals at different positions along the drift tube to set a series of reaction

21 durations.68b FA-SIFT is also a tandem-in-space MS for ion/neutral reactions with the reaction time in a well-defined fashion.

Figure 1.14 Schematic of flowing-afterglow selected-ion flow-tube (FA-SIFT) coupled with a radical source for ion/radical reactions69

In 2004, this instrument was involved in the study of ion/radical reactions by adding one additional radical source to the flow tube (Figure 1.14).69 The neutral radicals are generated by a pulsed pyrolysis nozzle (also known as Chen nozzle70). Pulsed pyrolysis has been an efficient means to produce gas phase radicals of high intensities in vacuum.71

It uses a silicon carbide tube for resistance-heating to dissociate organic precursors into neutral radicals for the structural study of highly reactive species using infrared or electronic spectra. Proton transfer reaction was observed between allyl radicals and hydroniums, also associative detachment reaction between ortho-benzynes and hydroxides, and radical recombination reaction between allyl radicals and benzyl radical cations.69 The results firmly showed that FA-SIFT is capable of carrying out ion/radical reactions with the additional radical source. No biological molecules have yet been studied using this specific MS instrument.

1.1.3.4 Fourier transform ion cyclotron resonance (FTICR)

Different from tandem-in-space mass spectrometers, mass analyzers such as Fourier transform ion cyclotron resonance (FTICR, Penning trap33) and quadrupole ion traps

(Paul trap72) are able to trap ions for a certain period of time, while carrying out multiple steps of reactions and mass analysis. The advantage of tandem-in-time mode is that the instruments do not require extra mass analyzers for performing additional stages of MS analysis or reactions. The tradeoffs, compared to tandem-in-space, are longer duty cycles and potential interference by the leftover neutral reactants.

FTICR employs a cyclotron cell that operates at a high vacuum of 10-7 to 10-8 Torr for trapping. It consists of three pairs of adjacent electrodes assembled into a cubic, cylindrical, or hyperbolic designs.73 A typical setup of FTICR is illustrated in Figure

1.15.74 The front and back electrodes, perpendicular to the strong magnetic field, function as trapping plates to trap the ions inside the cyclotron cell. A radio frequency (rf) is applied on the side plates to excite the trapped ions to induce the image current. The

detection plates (top and bottom) are used for monitoring the current induced by the ions 22

motions. A chirp is applied by sweeping the rf sweep from several kHz to several mHz to excite the trapped ions. The induced image current is collected in the time domain. Mass spectrum in frequency domain is then obtained with a fast Fourier transformation. The mass resolution on FTICR is correlated to the strength of the magnetic field so that accurate mass (< 1 ppm) can be obtained with large magnetic field (10T).

Figure 1.15 Schematic of a basic ICR cell, consisting of two trapping plates (front and back), two opposite excitation plates (right and left), and two detection plates (top and bottom). Ions enter the cell through the front trapping plate. Mass spectrum generated by fast Fourier transformation.74

Similar to SIFT, FTICR was used in the study of interstellar chemistry at very low temperatures68a and has been coupled with a pyrolysis nozzle.75 The reactions between benzyl radical cations and pyrolysis-produced allyl radicals were studied, which formed a carbon-carbon covalent bond. In another study, the protonated desR1-bradykinin ions were mass-selectively isolated and underwent a hydrogen/deuterium (H/D) exchange

23 reaction with deuterium radicals generated by electron gun, which was one of the very few reactions involving bio-ions and neutral radicals.76

Instead of carrying out reactions between bio-ions and neutral radicals, organic radical ions can be generated in gas phase, trapped in FTICR, and then reacted with gas- phase biomolecules. With laser-induced acoustic desorption (LIAD) techniques (Figure

1.16), neutral biomolecules, such as amino acids and peptide, can be made into gas phase and interact with the trapped radical ions.77 Abstraction, addition, and proton-transfer reactions have been observed for reactions between radical ions and biomolecules.

Figure 1.16 Schematic of the FT-ICR instrument, including the LIAD probe.78

Besides ion/neutral (ion/molecule, ion/radical) reactions, FTICR is also well-used in proteomic study, especially with the development of electron capture dissociation (ECD) by McLafferty and coworkers in 1998.16, 79 To apply this technique, trapped positive precursor bio-ions capture low energy electrons (<0.2 eV), forming odd-electron ions via ion/electron reaction.80 Charge reduced ion [M+nH](n-1)+• is the main product of electron capture, which causes peptide backbone cleavages.81 In contrast to CID, in which the weakest bond is cleaved through an ergodic process, the major cleavage occurs at N-Cɑ bond with ECD, resulting in c/z type of peptide ions.82 It is believed that an aminoketyl

24 • • radical is formed during the ECD, which subsequently produces c and z fragments (or c and z) as shown in Figure 1.17.83 The reaction is fast, regarded as non-ergodic and a perfect match with FTICR due to the low pressure in the cyclotron cell which allows efficient electron capture.16

Figure 1.17 Aminoketyl radicals and c and z• ions by ECD2, 84

The c/z pair of ions is unique in radical peptide ion fragmentation compared to even- electron species. One important feature of ECD is that the cleavages randomly occur, without a preferred site except for the proline amino cleavage.85 Thus, almost all the N-

Cɑ bonds throughout the backbone of peptides could cleave to generate fragments, which produces meaningful information for peptide and protein sequencing (Figure 1.18).86

Together with slow heating ion activation techniques in FTICR, such as sustained off- resonance irradiation collision-induced dissociation (SORI-CID), infrared multiphoton dissociation (IRMPD), and blackbody infrared radiative dissociation (BIRD),87 more internal energy is directed into the biological radical ions, and therefore more fragments can be produced for proteins over 20 kDa. On the other hand, posttranslational modifications can be preserved using ECD, which makes it possible to identify disulfide linkage, glycosylation, phosphorylation and oxidation of proteins.2, 88

Figure 1.18 Mass spectrum of ubiquitin ([M+9H]9+) by ECD FTICR with 9.4 T instrument.89

A variety of gas-phase reactions, including ion/molecule, ion/radical, and ion/electron reactions, have been investigated using FTICR-MS to study the biological molecules. FTICR-MS instruments with a large magnetic field are of high cost for ownership and maintenance,45 in addition, the number density of neutral reactants is relatively low due to the high vacuum requirements, which is not in favor of ion/molecule or ion/radical reactions.

1.1.3.5 Quadrupole ion traps

Quadrupole ion traps are operated with radio-frequency (rf), which creates a quadrupole electric field to trap the ions.72 Based on different geometries, quadrupole ion traps can be categorized into 3D ion traps, and linear or 2D ion traps.72 The linear ion traps (LITs) have increased trapping capacity. Quadrupole ion traps are effective tools for in-vacuum gas-phase ion reactions because of their capability to store ions for long reaction time (as high as 10 seconds) and the relatively higher pressure reaction

-3 -5 26

environment (10 -10 Torr). Since gas-phase reactions occur via collisions, which is highly dependent on the number density, the pressure inside quadrupole ion traps is three orders of magnitude higher than FTICR, making them highly preferable for carrying out gas-phase ion reactions. With these features, quadrupole ion traps, especially linear ion traps, have been widely employed for studies of gas-phase ion reactions.

Neutral reagent vapor can be simply mixed with the collision gas for ion/molecule reactions in the ion trap. An example is shown in Figure 1.19a. The neutral reactants were leaked into Q3 linear ion trap of a modified QTrap 4000 (AB Sciex) mass spectrometer to

27 investigate the reactivity of thiyl, sulfinyl, and perthiyl radicals.42 To introduce multiple neutral reagents subsequently into ion trap mass spectrometers, a multi-valve interface consisting of three neutral lines was designed,90 where three different ion/molecule reactions could be carried out at the same time for fast neutral reagent screening (Figure

1.19b).

Figure 1.19 Schematic of a single neutral injection line (a)42 and a multiported pulsed valve interface (b)90 of a linear quadrupole ion trap for ion/molecule reactions.

As one of tandem-in-time mass spectrometers, ions are manipulated in one trap mass analyzer to achieve multiple stages of mass analysis. For target analyte analysis, MSn with CID can be easily carried out via isolating the precursor ions followed by multi- collisional dissociation (ac activation induced rf heating). However, the structural identification for the products of ion/molecule reactions can be tricky since neutral

28 reactants remain inside the ion traps and can further react with the fragment ions. One solution to this issue is to introduce two linear quadrupole ion traps in series as shown in

Figure 1.20,91 which borrows the idea of tandem-in-space MS systems yet keeps the advantages of tandem-in-time MS systems. Ion/molecule reactions were carried out in the first trap (LQIT1); the product ions of interest were then transferred into the second trap

(LQIT2) for structural determination; or vice versa, the CID products from LQIT1 could be transferred to LQIT2 for ion/molecule reactions.

Figure 1.20 Schematic of the differentially pumped dual LQIT with ion optics and differential pumping. LQIT1 and LQIT2 are the ﬁrst and second linear quadrupole ion traps (LQIT).91

Gas-phase ion/ion reactions usually require longer reaction time and high abundance for both positive and negative ions, and one of them needs to be multiply charged for the 28

reaction products to be analyzed by MS.92 Ion/ion reactions can be readily applied to peptides and proteins since multiply charged ionic species can be produced through spray-based ionization methods (ESI or nanoESI). Electron transfer dissociation (ETD), proven to be one of the most useful applications of ion/ion interactions, occurs between peptide or protein cations and radical anions in ion traps. Ion/ion reactions take place at the millisecond level, much faster than ECD, which is limited by the slow electron transferring rate. Thereby, ETD can be coupled with liquid chromatography.93 Reagents such as anthracene and azobenzene are used to generate radical anions by chemical

29 ionization (CI) or atmospheric pressure chemical ionization (APCI).94 These anions act as electron donors due to their low electron affinity. Through electron transfer, the odd- electron ions of reduced charge are produced and fragmented into c/y pairs from peptide ions. A competitive proton-transfer reaction also occurs in ETD,95 giving even-electron ions by losing a proton similar to the products in ECD through hydrogen loss. The advantages of ETD include its low cost, the use of quadrupole ion traps instead of FTICR, and the flexibility in ion manipulation. Low energy CID of all the resulting ions from electron transfer reaction (supplemental collisional activation) or one specific charge- reduced species (charge-reduced species CID) can be carried out with high sequencing coverage.45, 96 Instrumentation of ion/ion reactions is generally more complicated, compared to those for ion/molecule reactions, since two different ion beams are required.

Unlike 2D ion traps, 3D ion traps can store ions of both polarities.92, 97 As shown in

Figure 1.21, a home-built 3D ion trap instrument was constructed with three ion sources: a positive and a negative ESI sources to generate multiply charged peptide/protein ions,

and an atmospheric sampling glow discharge ionization (ASGDI) source to produce ions 29

of either polarity for reactions.97-98 The turning dc quadrupole was used for selecting the ion beam into the 3D ion trap through the holes on the endcap, while the ASGDI source was directly pointing at the hole of the ring electrode. Studies of both proton-transfer and electron transfer reactions were carried out using this configuration.

Figure 1.21 Schematic of a multi-source quadrupole ion trap instrument containing two electrospray ionization (ESI) sources and one atmospheric sampling glow discharge ionization (ASGDI) source interfaced.98b

Linear (2D) ion traps have inherent advantages over 3D ion traps, such as higher trapping capacity and higher trapping efficiency for externally injected ions, mutual storage of ions of opposite polarities is not effective in conventional instruments92. Two approaches have been reported for mutually trapping cations and anions: (1) applying rf 30

directly to at the end electrodes of the linear ion trap to create rf trapping along z-axis,17 so that positive and negative ions can be axially trapped simultaneously for reaction and

(2) using an unbalanced trapping rf to create a trapping field along the axial direction.99

Ion/ion reactions can also be obtained in transmission mode, whereby ions of one polarity are trapped in the linear ion trap, and ions of the other polarity are injected through the ion trap for reaction. An example is shown in Figure 1.22,100 the negative ions generated by ASGDI were injected through Q3 and accumulated in Q2 before multiply charged positive ions were injected through Q2, and the final products were analyzed in Q3. The

31 radial injection of negative reactant ions into Q3and subsequent trapping in Q2 is not efficient, which limits the effectiveness of the transmission mode. A pulsed dual ion source consisting of +ESI/-ESI or ESI/APCI has been utilized to generate and introduce ions of opposite polarities subsequently into Q2 linear ion trap for reaction through the same ion pathway but different potentials on the optics.101 Proton-transfer and ETD reactions have been investigated by employing the pulsed dual ion source.

Figure 1.22 Schematic of a triple quadrupole/linear ion trap modified by addition of an atmospheric sampling glow discharge ionization source on the side of Q3 linear ion trap for ion/ion reactions.100 31

Quadrupole ion traps are also feasible for studying ion reactions with metastable species and photons based on specific modifications. Two modified instruments used in these studies are shown in Figure 1.23. A pulsed fast atom bombardment was placed next to a hole on the ring electrode of a 3D ion trap to generate metastable atoms to react with peptide ions for inducing dissociation;19 laser emission was directed into a linear ion trap via optics for peptide ion/photon reactions. Metastable atom-activated dissociation

(MAD),19 infrared multiple photon dissociation (IRMPD),102 and ultraviolet photon

32 dissociation (UVPD)102a, 103 have been developed using the interaction of peptide ions with metastable species, infrared, and ultraviolet photons, respectively, to produce odd- electron ions for peptide sequencing. Ion/neutral radical reactions should also be employed in quadrupole ion trap mass spectrometers if additional radical sources are installed. The application of gas-phase ion/radical reactions in an ion trap mass spectrometer is still under investigation.

Figure 1.23 Schematic of (a) a 3D ion trap coupled with a fast atom bombardment (FAB) gun for metastable atom-activation dissociation19 and (b) a laser coupled linear ion trap for infrared multiple photon dissociation (IRMPD)102b and ultraviolet photon dissociation (UVPD)103.

1.1.3.6 Hybrid instruments

Linear quadrupole ion traps have large trapping capacity and allow axial ion injection, which makes them particularly attractive reactors for gas-phase ion reactions in hybrid instruments. High accuracy mass analyzers (FTICR, TOF and Orbitrap) can be coupled with linear ion traps to study a wide variety of gas-phase ion reactions. In this section, two hybrid instruments, one with a TOF analyzer and one with an Orbitrap analyzer are presented as examples of hybrid instruments for gas-phase ion reactions.

Quadrupole tandem time-of-flight (QTOF)

Figure 1.24 shows a quadruople tandem time-of-flight (QTOF) mass spectrometer modified for ion/ion reactions.104 This instrument contained three quadrupoles (ion guide

Q0, mass filter Q1, collision cell Q2 as a linear ion trap) and an orthogonal reflectron

TOF analyzer for product analysis. A pair of pulsed ion source, ±ESI or nano-ESI

(+)/APCI (-), were placed in front of the MS inlet to produce the ionic reactants of

opposite polarities to study proton transfer reactions or ETD reactions. The reflectron 33

TOF significantly had improved mass resolving power and accuracy, compared to quadrupole mass filter or Paul traps. The mass resolving power is about 8000 over a wide mass range up to m/z 6,600 with a mass accuracy better than 20 ppm.

Figure 1.24 Schematic of a modified quadrupole time-of-flight tandem mass spectrometer for ion/ion reactions with dual ion source.104

Orbitrap Instruments

Orbitrap is a mass analyzer for exact mass measurement, developed by Makarov and coworkers based on a King trap.105 The ions are trapped by the electrostatic field with a

centrifugal forces. Image current is measured for the harmonic axial motions. Hybrid 34

linear ion trap tandem Orbitrap (LTQ-Orbitrap) was commercialized in 2005.106 The recent Orbitrap Elite (Figure 1.25)107 consists of two linear ion traps with different pressures,108 a quadrupole mass filter, a C-trap, a collision cell and an Orbitrap mass analyzer with an optional ETD reagent generator. The high-pressure ion trap is used to cool down the ions whereas the low-pressure cell is for mass analysis or isolation. Mass- selected precursor ions can be transferred along the quadrupole mass filter to the C-trap.

C-trap is used to cool and transfer ions into the Orbitrap. In the early design, C-trap was also used to carry out slow-heating CID by increasing the rf, but with the restriction of

35 low-mass cutoff (LMCO) through the rf ramping.109 Later on, another linear ion trap

(HCD collision cell) is used for high energy CID. Negative ETD reagent ions can be introduced through ETD cassette into the HCD collisional cell as well. Gas-phase ion reaction products are then transferred back to the C-trap before entering Orbitrap for accurate mass measurement. Hybrid Orbitrap mass spectrometers have been used intensively for the analysis of protein, glycan and biopharmaceutical applications.

Figure 1.25 Schematic of an Orbitrap Elite.107a

1.1.3.7 DAPI mass spectrometers

For the study of gas-phase ion reactions in hybrid instruments, more delicate 35

compartments can be installed to achieve multi-functions of high performance and high compatibility, while compartments can also be reduced and simplified as long as the basic requirements of in-vacuum ion reactions are satisfied. In all MS instruments mentioned in the previous sections with atmospheric ion sources such as ESI and APCI, the continuous atmospheric pressure interface (API) requires differential pumping system to support the high vacuum for mass analysis as well as ion optical tools to guide the ions into the analyzers. Discontinuous atmospheric pressure interface (DAPI),110 on the other hand, is another solution to substitute for the massive interface (removing all ion optics)

36 and differential pumping (largely reducing pumping requirements) in the conventional

MS instruments as shown in Figure 1.26. The original purpose of using DAPI together with a rectilinear ion trap (RIT)111 was to miniaturize the mass spectrometers.112

Figure 1.26 Schematic of (a) conventional linear ion trap mass spectrometer with differential pumping system and ion optics and (b) miniature DAPI-RIT mass spectrometers highlighted Mini 11 mass spectrometer of 9 pounds and Mini 12 of Point- of-care personal mass spectrometer.110, 113

The operation of a DAPI-MS consists of four steps (Figure 1.27a):110 (1) ions are

generated in the atmosphere by nanoESI or APCI before the DAPI opens; (2) the pinch valve in DAPI opens for 10 to 30 milliseconds to transfer the ions into the trap in vacuum chamber. In this process, rf is applied to trap ions and the pressure inside chamber increases to 10-2-10-1 Torr; (3) after ion introduction, the DAPI closes and the ions stored in the trap cool down for hundreds of milliseconds while the pressure decreases to 10-3

Torr; (4) at this pressure, mass-selective instability scan is used for mass analysis. One scan cycle of a DAPI mass spectrometer usually takes one second, as the tradeoff for breaking the barrier of pumping speed to not include a differential pumping system.

Figure 1.27 (a) Scan function for mass analysis using a DAPI mass spectrometer. (b) Manifold pressure measured during scanning, with an open time of 20 ms and a closed 110 time of 850 ms for DAPI. 37

Multiple DAPIs can be implemented to introduce charged or neutral species for gas phase ion reactions with minimum modification to an instrument. Note that the pressure during the ion or neutral introduction is different from that for mass analysis (Figure

1.27b). This represents a unique opportunity for using a single trap to carry out gas-phase reactions at a much elevated pressure, with reaction products later analyzed at 10-3 Torr in a single scan cycle. One previous study114 by our research group has shown that two beams of ions or neutrals can be introduced into a LIT sequentially or simultaneously

38 through two DAPIs for gas phase ion processing, including gas flow CID, gas flow assisted desolvation, and one ion/ion reaction. Simplified home-built mass spectrometer with multiple DAPIs could be potentially constructed to investigate a variety of gas-phase ion reactions, especially the interactions between biological ion and organic radicals, with additional radical sources like the pyrolysis valve.71

1.2 Conclusion

Gas-phase reactions of biological ions especially those producing odd-electron bio- radical ions, have been applied for biomolecule structural identification, disease diagnosis, and enzymatic reactivity evaluation. Gas-phase bio-ion interactions can be carried out in atmosphere or in vacuum. Significant basic chemistry and applications have been shown in the study of gas-phase biological ion/radical reactions in the ESI plume or bulk solution. Minimum instrumentational modification is required for such reactions at atmospheric pressure, however no single ionic reactant can be selected prior to the

reactions. Ion manipulation and mass-selectively isolation can be achieved for gas-phase 38

ion reactions in vacuum, which on the other hand, require multi-stages tandem mass spectrometry. Various tandem mass spectrometers of both tandem-in-space and tandem- in-time configurations, have been demonstrated as useful tools for studying in-vacuum ion/molecule, ion/ion, ion/radical, and ion/photon reactions. Among all the mass analyzers, ion traps are feasible for all kinds of reaction types with long ion storage time.

Quadrupole linear ion traps are ideal reaction vessels for gas-phase reactions due to the convenience of introducing ionic or other reactants and the relatively high pressure for reactions. Hybrid instruments employ linear ion traps as the reactor and accurate mass

39 analyzers for product analysis, which represents an effective way of improving the instrumentational performance in studying gas-phase reactions. On the other hand, a multi-DAPI mass spectrometer presents an opposite approach that is “just good enough” for the study, with the simplest means to introduce reactants in a controlled environment.

This thesis features a DAPI-RIT-DAPI mass spectrometer that has been developed with a rectilinear ion trap as both the reactor and the mass analyzer, and two pulsed interfaces to introduce ions and other species for reactions. Ion/molecule and ion/ion reactions were used to characterize the established system at an elevated reaction pressure and with the energetic gas flow by the DAPIs. With an additional radical source, a novel ion/radical reaction between protonated alkyl amines and pyrolysis-generated C3H2 carbene bi-radicals to form a C-N covalent bond was discovered with the instrumentational setup shown in Figure 1.28. This reaction was further applied to biological ions such as nucleobases, nucleosides, amino acids, peptides, and proteins.

Figure 1.28 Schematic of a DAPI-RIT-DAPI mass spectrometer coupled with a pyrolysis radical source for the study of gas-phase ion/radical reactions.

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CHAPTER 2. INSTRUMENTATION AND CHARACTERIZATION OF A HOME- BUILT DAPI-RIT-DAPI MASS SPECTROMETER FOR GAS-PHASE ION/MOLECULE AND ION/ION REACTIONS

2.1 Introduction

Mass spectrometry (MS) has been widely used to study gas-phase ion chemistry via inducing interactions or reactions between ions and matter in vacuo, such as neutrals,1 radicals,2 electrons,1c, 3 ions,4 photons,5 surfaces,6 metastable species,7 etc. Besides being used as a mass analyzer, a quadruple ion trap can serve as a reactor for studying gas- phase reactions with capabilities of manipulating the trapped ions in a controlled-time fashion at relatively high pressure for reactions. Owing to the development of MS systems with atmospheric pressure ionization sources such as electrospray ionization

(ESI)8 and atmospheric pressure chemical ionization (APCI)9, ions of intact molecules can be generated directly from condensed phase at atmospheric pressure. The 50

atmospheric pressure interface (API) is an essential component in these mass spectrometers, through which the ions are transferred from atmosphere to the mass analyzer in the vacuum.

The APIs adopted in commercial mass spectrometers typically perform at continuous ionization and ion introduction mode. The continuous APIs require a differential pumping system with a large pumping capacity to support multiple vacuum regions at different pressures. Delicate ion optics is used to transfer the ions through different regions.10 Instrumentational modification is always needed to conduct gas-

51 phase reactions on a mass spectrometer consisting of a single API. For instance, neutral reactant needs to be leaked into the vacuum for ion/molecule reactions,11 which is practically done for quadrupole ion trap or filter instruments through the collision gas introduction line. To implement ion/ion reactions, a different ionization interface is typically required to introduce an ion beam of the other charge polarity,12 which can demand significant efforts in the instrument modification. An alternative solution is to use a discontinuous atmospheric pressure interface (DAPI) for introduction of ions or neutrals, which was initially developed for miniature mass spectrometers and has been demonstrated to be effective with small pumping systems.13 In a DAPI, a pinch valve is used to control the opening of the interface (10 to 30 ms) and the ions are transferred from the atmosphere directly to the ion trap mass analyzer in the vacuum. During the ion introduction period, the pressure inside the vacuum manifold increases to 10-2 to 10-1 Torr.

The ions are trapped at the elevated pressure and mass analyzed after a delay of several hundred milliseconds to allow the pressure dropping back to 10-3 Torr.13b DAPI can be

14 implemented even with bent capillaries without losing ion intensity significantly, which 51

provides flexibility for the instrument modification. It has been demonstrated in a previous study15 that multiple beams of ions or neutrals can be introduced into a rectilinear ion trap (RIT)16 sequentially or simultaneously through two DAPIs for gas phase processes including collisional induced dissociation, gas flow assisted desolvation, and ion/molecule or ion/ion reactions.

In this study, we adopted the simple DAPI-RIT-DAPI configuration previously proposed15 to set up an instrument that could potentially be used for gas-phase ion reactions with multiple beams of ions or neutrals conveniently introduced through the

52 two DAPIs. A distinct feature of the MS systems with DAPI is that the in-vacuum pressure varies during a scan cycle and the ions trapped in vacuum experience stronger and more energetic gas flows. This represents a unique opportunity for using a single trap to carry out gas-phase reactions at a condition different from MS analysis of the reaction products in a single scan cycle. The pressure changes and gas flow impact can be critical to gas-phase reactions and need to be characterized for studying complicated reactions.

Herein, the gas dynamic effects on ion internal energy and ion reactivity as well as the pressure effect on the reaction rate have been investigated. Two types of ion reactions were used to test this system, including ion/molecule and ion/ion reactions. Ion/ion reactions have been widely applied in proteomics1c and they have been made available on several types of commercial instruments. We are interested to test the performance of our instrument with extremely simplified ion optics for ion/ion reaction applications.

Ion/molecule reactions, on the other hand, offer a great means for studying gas-phase ion structures and energetics. Many research labs have modified their instruments to facilitate

this type of fundamental studies. In this work, we chose ion/molecule reactions involving 52

peptide radical ions because the structures and reactivity of radical ions are very sensitive to instrument conditions, e.g. energy deposition associated with gas collisions during the ion transfer and storage.17

2.2 Instrumentation

The home-built DAPI-RIT-DAPI mass spectrometer was developed using the vacuum chamber with inside dimensions of 35 × 25 × 25 cm. The manifold was pumped by a 210 L/s turbo pump (THH 262 P, Pfeiffer Vacuum Inc, New Hampshire) and a 30

53 m3/h rotary vane pump (UNO-030M, Pfeiffer Vacuum Inc., New Hampshire). A nanoESI emitter consisting of a tip-pulled (i.d. < 20 m) boron silica glass capillary (0.85 mm i.d. and 1.5 mm o.d.) was used to generate ions in front of DAPI-1 (the left DAPI in Figure

2.1a). Other reagents were introduced by the gas flow through DAPI-2. A pinch valve

(ASCO 390NC24330, ASCO Scientific, Florham Park, NJ) with a semi-conductive silicone tubing (3 cm long, ∼350 Ω resistance, with 1.6 mm i.d. and 3.2 mm o.d.) was used for each DAPI. Capillary 1 (10 cm long and 0.5 mm i.d.) was attached to DAPI-1;

Capillary 2 (25 cm long and 0.75 mm i.d.) and Capillary 3 (5 cm long with various i.d.s tested) were attached to DAPI-2. All the capillaries were grounded. The DAPI-1 and 2 were controlled separately each by a 0-24 V pulsed dc signal from the control electronics.

An RIT with dimensions x0 = 5 mm, y0 = 4 mm, and z = 40 mm was operated with a single-phase radiofrequency (rf) at 850 kHz for ion storage and MS analysis. The end electrodes were made from electrogalvanized steel mesh electrodes (0.009” Wire,

McMaster-Carr, Aurora, OH) approximately 2 mm away from the capillaries of each

18 DAPI to allow ion/neutral injection. A previous study with simulations has shown that a 53

mesh electrode, in comparison with a plate electrode with a center hole, can minimize the disturbance to the trapped ions by the gas flow from the DAPI while allowing high throughput of the ions. A set of control electronics, from a Griffin Minotaur 300 (FLIR

Systems, Inc., Wilsonville, OR), was used for the instrument control. A 30 V dc was applied on both end electrodes to create a dc trapping potential well along the z direction.

Resonance ejection was implemented for MS scan with a dipolar ac signal at 300 kHz (q

= 0.81). The conversion dynode of the detector was set to 0 V for positive ion mode and

+4000 V for negative ion mode. The isolation of the trapped ions was achieved by a

54 notched SWIFT (stored waveform inverse Fourier transform). Collision-induced dissociation (CID) was implemented by dipolar excitation with a single-frequency ac applied between the x electrodes of the RIT. The typical nanoESI spray voltage was set to

+/-1600 V in positive/negative ionization mode. Multiple triggers were provided from the control electronics for synchronizing the operations of nanoESI, DAPIs, and MS analysis.

All the spectra presented are the average of three continuous scans.

2.3 Materials

S-nitrosoglutathione (γ-E(NOC)G), dimethyl disulfide, allyl iodide, angiotensin I

(single letter sequence: DRVYIHPFHL), pentachlorophenol (PCP), 9-anthracene carboxylic acid, cocaine, and benzylpyridinium thermometer ions were purchased from

Sigma Chemical Co. (Sigma-Aldrich, St. Louis, MO). Different concentrations (5-100

μM) of the samples were prepared with methanol/water (1:1, v/v) with 0.2 % acetic acid added to increase the intensity of protonated species if necessary.

2.4 Results and discussion

2.4.1 System configuration

The DAPI-RIT-DAPI system (Figure 2.1a) consists of two DAPIs, one RIT, and one detector. The general scan function for gas-phase reactions shown in Figure 2.1b was set as following. DAPI-1 was opened for 10-30 ms to introduce ions into the RIT; the ions of interest for reaction were isolated using SWIFT, and CID was performed and followed by another isolation if a fragment ion was used for reactions; the second DAPI, DAPI-2 was opened to introduce other reagents into the RIT, which can be neutral molecules for

55 ion/molecule reactions or ions for ion/ion reactions. Capillary 3 could be easily switched with capillaries of different inner diameters from 0.125 to 0.75 mm, which was selected based on the desirable pressure or reaction time (for neutrals) for the reaction. An opening time as long as 250 ms could be applied with a 5 cm capillary of id 0.125 mm and multiple introductions could also be implemented.15 The species introduced from both DAPIs interact with each other inside the RIT during the cooling period after DAPI-

2 was closed. The final product ions were analyzed by rf scan with resonance ejection at q = 0.81. CID could also be performed to the product ions.

Figure 2.1 (a) Schematic representation of the DAPI-RIT-DAPI system; (b) General waveforms of the in-trap gas-phase reactions

Two types of reactions, ion/molecule and ion/ion reactions, were performed using the DAPI-RIT-DAPI system to show the feasibility of this configuration. The product spectra are shown in Figure 2.2 and Figure 2.3a, respectively. For ion/molecule reactions, peptide thiyl radical ions [γ-E(S•C)G+H]+ (m/z 307) were generated by CID19 of the protonated S-nitrosoglutathione [γ-E(NOC)G+H]+ (m/z 337) introduced through DAPI-1

(Scheme 2.1) and reacted with the allyl iodide vapor introduced through DAPI-2

(capillary 3: i.d. 0.25 mm, opening time 25 ms). The reaction time was set to 750 ms after closing DAPI-2 and before rf scan. The thiyl radical located on the cysteine side-chain attacked the C-I bond within allyl iodide, leading to the formation of allyl adduct at m/z

348 (+•C3H5, +41 Da) and iodine adduct at m/z 434 (+•I, +127 Da). The reactions between the thiyl radical with dimethyl disulfide was also observed with product ions at m/z 354 (+•SCH3, +47 Da). The reaction phenomenon is consistent with literature reports on peptide thiyl radical species (Scheme 2.2).20 The details of the thiyl radical reaction will be further discussed later to characterize the gas flow effect on the radical reactivity.

Figure 2.2 Ion/molecule reactions: thiyl radical cations react with allyl iodide

Scheme 2.1

Scheme 2.2

For ion/ion reactions, protonated angiotensin I (DRVYIHPFHL) generated via positive mode nanoESI (77 μM) was introduced into the RIT through DAPI-1 with a pulsed open time of 30 ms (Figure 2.3b). The triply charged ions (m/z 433) were isolated at q = 0.80 after 250 ms of cooling time (Figure 2.3a inset). The anion reagent was generated with negative mode nanoESI of 37 μM pentachlorophenol (PCP, MW: 266.34) and introduced through DAPI-2 with capillary 3 of 0.25 mm i.d. for 60 ms. The single- phase rf applied on the RIT allowed efficient trapping of both cations and anions with the dc voltage on the end electrodes set to zero. A low-mass cutoff at m/z 220 and a reaction time of 260 ms were used (scan function shown in Figure 2.3d). As shown in Figure 2.3a, 57

product ions resulted from proton transfer reactions, i.e., doubly charged angiotensin I at

+ + 21 m/z 649, can be well detected. Noticing that fragment ions such as b4 and y4 were observed, which was not common in ion/ion reactions. This phenomenon is probably due to an impact of the second gas flow (collisional activation) on the triply charged peptide ions. Same fragments were also observed when using the same scan function (DAPI-2 opened), however, with the nanoESI source turned off for anion production (Figure 2.3c).

Figure 2.3 (a) Ion/ion reactions: Angiotensin I triply charged cations react with deprotonated pentachlorophenol anions (insert: Isolation of the triply charged species); (b) MS spectrum of Angiotensin I without opening DAPI-2; (c) MS spectrum of isolated triply charged peptide ions with DAPI-2 opening but the nanoESI source being turned off for anion production; and (d) aveforms of the ion/ion reaction (dc was set to -10 V during mass analysis).

Based on the results of Figure 2.2 and Figure 2.3, both ion/molecule and ion/ion

reactions could be performed on the DAPI-RIT-DAPI system, while the pressure change and the energy deposition by the gas flow occur during the DAPI opening. As discussed above, these two features could play important roles in gas-phase reactions. In the following studies, we focused on the characterization of these two factors for carrying on the gas-phase ion reactions using the DAPI-RIT-DAPI system.

2.4.2 Pressure effect on gas-phase ion reactions

With the DAPI MS systems, the pressure in the ion trap can rise to 10-1 Torr or higher after the opening of DAPIs, which is much higher than the pressure for conventional MS analysis using quadrupole ion traps (mTorr or lower). After the DAPI is closed, the pressure is gradually pumped down. By varying the delay time for performing the CID, the dissociation reactions can be carried at different pressures. As shown in a series of CID experiments (q = 0.30) of cocaine ([M+H]+, m/z 304, 3 μM, Scheme 2.3) at different trap pressures, the optimal fragmentation efficiency (defined as the fraction of fragment ion intensity over total ion intensity) occurs at ~8 x 10-4 Torr as shown in Figure

2.4. The activation time was set to 30 ms and the amplitude of the excitation AC was 200 mV. The relatively lower fragmentation efficiency at pressures lower than 5 x 10-4 Torr is likely due to the lower collision rate at lower pressures. The decrease in fragmentation efficiencies at pressures higher than 3 mTorr could be due to the significantly enhanced damping effect that cools the ions to the center of the trap.22

Scheme 2.3

Figure 2.4 CID spectra and MS/MS efficiency of protonated cocaine m/z 304 at different pressures. Activation time 30 ms, amplitude 200 mV.

CID was also performed (q = 0.30) for deprotonated 9-anthracene carboxylic acid

(m/z 221, 5 μM) at different pressures (Figure 2.5). When the CID was performed at about 6 mTorr (200 ms after DAPI-1 opening), only product ion m/z 195 was observed

(Figure 2.5a); At pressure of about 2 mTorr (500 ms delay), another product ion m/z 177 was observed in addition to m/z 195 (Figure 2.5b); Product ion m/z 177 became the only product when CID was carried out at 1 mTorr or lower (780 ms delay) (Figure 2.5c). The ions m/z 177 could be obtained also by isolating and activating m/z 195 (Figure 2.5d).

The CID activation time was 30 ms. In-trap CID experiments were performed on a commercial triple quadrupole/linear ion trap (QTRAP 4000, AB Sciex, Toronto, Canada) for product verification. The precursor ions at m/z 221 were isolated in Q1 mass filter and fragmented in Q3 linear ion trap via on-resonance CID. As shown in Figure 2.5e, the only fragment pathway observed was CO2 loss, giving rise to the anthracene anion at m/z 177, which has been demonstrated as an ETD reagent.23 This result only matches the CID results obtained at low pressure when using the DAPI instrument (Figure 2.5c). Given the high reactivity of anthracene radical anions, m/z 195 observed under higher pressures

(Figure 2.5a and b) might result from sequential ion/molecule reactions of m/z 177 with trace H2O in the trap. To test this hypothesis, water vapor was mixed into the collision

gas on QTRAP 4000. The anthracene anions (m/z 177) were stored in Q3 linear ion trap 61

-5 for various reaction times with H2O at the trap pressure of 4 × 10 Torr. Figure 2.5f shows the data after 3 s reaction time, in which m/z 195 can be clearly detected. A peak at m/z 221 was also observed, which is probably due to reaction of the anthracene anions with trace CO2 in the water vapor. Note that the reaction time on QTRAP 4000 was long

(3000 ms), yet anthracene anions at m/z 177 remained as the base peak, while 100% yield of water adduct was observed with the DAPI instrument at only 600 ms reaction time

(Figure 2.5a).

Figure 2.5 Ion-trap CID spectra of deprotonated 9-anthracenecarboxylic acid at (a) 6 mTorr 200 ms delay, (b) 2 mTorr 500 ms delay, (c) 1 mTorr 780 ms delay, from the DAPI opening on DAPI-RIT-DAPI system. The yellow, red, and blue columns illustrate the positions of ion introduction, dipolar ac excitation, and MS scan, respectively; (d) Ion-trap CID spectrum of m/z 195 on DAPI-RIT-DAPI system; (e) Ion-trap CID spectrum of deprotonated 9-anthracenecarboxylic acid on QTRAP 4000; (f) Ion/molecule reactions of deprotonated anthracene ions with water vapor on QTRAP 4000. The CID activation time was set to 30 ms on DAPI-RIT-DAPI system.

As a summary, an optimal CID efficiency was obtained at about 0.8 mTorr and ion/molecule reactions happened at much faster rates at higher pressures (6 mTorr) using the DAPI-RIT-DAPI instrument and therefore the reaction products can be obtained with

short reaction time.

2.4.3 Dynamic gas flow effect on gas-phase ion reactions

According to the previous simulation study,18 the gas expansion after a DAPI is significantly larger and the gas velocity is much higher than those for a continuous API.

To characterize the internal energy distributions of the ions via DAPI introduction, the

“survival yield” method, which has been developed to determine ion internal energies,24 was applied in our study. A comparison was made between the DAPI using the home-

63 built instrument and a continuous API using the QTRAP 4000. A methanol/water (1:1, v/v) solution containing the following five compounds at 10-4 M each, p- chlorobenzylpyridinium chloride (p-Cl), p-methylbenzylpyridinium bromide (p-CH3), p- methoxybenzylpyridinium tetrafluoroborate (p-OCH3), p-nitrobenzylpyridinium bromide

(p-NO2) and p-cyanobenzylpyridinium chloride (p-CN), was used to generate the thermometer ions by nanoESI. The ions were introduced into the RIT directly through

DAPI-1 (DAPI opening time of 15 ms with a low-mass cutoff m/z 60) in the home-built instrument, or through the continuous API into Q3 linear ion trap of QTRAP 4000 with declustering potential of 10 V, collisional energy of 5 V, and Q3 entry barrier of 2 V (the minimum energy setup on the commercial instrument), respectively. Figure 2.6a shows the breakdown curves that were calculated and plotted using the methods described previously.24b In detail, the critical dissociation energies (E) of the five benzylpyridinium ions (p-OCH3, p-CH3, p-Cl, p-CN, and p-NO2) are 1.51, 1.77, 1.90, 2.10, and 2.35 eV, respectively. The survival yield (SY) was calculated as I(parent ion)/[ I(parent ion) + Σ

I(fragment ion)], where I(parent ion) and I(fragment ion) represent the intensities of 63

precursor and fragment ions, respectively. The corresponding internal energy distribution

P(E) (Figure 2.6b) was then calculated by taking the derivative of the breakdown curve in

Figure 2.6a. The internal energy distribution associated with DAPI is about 0.4 eV higher than the continuous API.

Figure 2.6 Energy characterization of DAPI. (a) Calculated survival ion yields and (b) Internal energy distributions of DAPI vs. continuous API for ion introduction;

Fragmentation spectra of thermometer ions after opening DAPI-2 for 20 ms with 64 capillary 3 (c) i.d. 0.5 mm and (d) i.d. 0.75 mm. *p-R represents the fragment of p-R (R = OCH3, CH3, Cl, CN, NO2).

To conduct reactions on the DAPI-RIT-DAPI instrument, a second reagent species was introduced with a gas flow from DAPI-2 after the ions injected through DAPI-1 had been trapped and cooled. It was observed that the second gas flow could cause the dissociation of the ions of small organic compounds from our previous studies15 and triply charged peptide ions. Therefore, the ion internal energy change due to the second gas flow was investigated using the thermometer ions. After the thermometer ions were

65 introduced through DAPI-1, their corresponding molecular ions were isolated and the

DAPI-2 was opened while an rf voltage corresponding to a low-mass cutoff of m/z 60 was used. No fragmentation of the thermometer ions was observed with capillary 3 of

0.125 mm or 0.25 mm i.d., when DAPI-2 was open for up to 250 ms. The most fragile p-

OCH3 ions fragmented with a capillary 3 of 0.5 mm i.d. and an opening time of 20 ms

(Figure 2.6c), while p-OCH3, p-CH3, and p-Cl started to fragment with capillary 3 of 0.75 mm i.d. DAPI-2 opening time 20 ms (Figure 2.6d). The disturbance and the energy deposition to the trapped ions could be controlled by selecting capillary 3 of proper conductance, i.e. capillary i.d. smaller than 0.5 mm. However, for introduction of ions for a reaction, the transfer efficiency decreases significantly when the capillary i.d. is smaller than 0.25 mm.13b These results indicate that the second gas flow led to an energy deposition to the trapped ions. Its influence on the reactivity of the radical ions was further studied with the reactions between glutathione thiyl radical ions and dimethyl disulfide.

As discussed earlier, glutathione thiyl radical ions could be produced from 65

protonated S-nitrosoglutathione (30 μM) through CID by a facile NO loss (Scheme 1).19,

20b Figure 2.7a shows a MS spectrum with a DAPI-1 opening time of 15 ms using a scan without applying excitation AC for CID. A fragment ion at m/z 307 was observed, corresponding to the loss of a NO from the precursor (m/z 337). This ion presumably is due to additional internal energy deposition to the precursor during ion introduction through DAPI-1, for which the average internal energy distribution is 0.4 eV higher than that from continuous API as shown in Figure 2.7b. For the ion/molecule reaction, the protonated S-nitrosoglutathione (m/z 337) was isolated and excited to produce thiyl

66 radical ions (m/z 307) through CID (Figure 2.7b) for subsequent ion/molecule reactions.

The head-space vapor of dimethyl disulfide (placed in a glass tube with a plastic

Swagelok connected to capillary 3) was introduced into the vacuum chamber for reaction through DAPI-2. Dissociative addition of the dimethyl disulfide to thiyl radical was observed to occur (Figure 2.7c), leading to the formation of glutathione methyl disulfide

(Scheme 2) at m/z 354. The reaction yield, defined as the fraction of the product intensity

(m/z 354) over the total ion intensity (sum of m/z 307 and m/z 354), was 70% under this condition.

Figure 2.7 Ion/molecule reactions of peptide thiyl radical ions and dimethyl disulfide. (a) MS spectrum of S-nitrosoglutathione; (b) Thiyl radical (m/z 307) produced by CID of protonated S-nitrosoglutathione; (c) Reactions between thiyl radicals and dimethyl disulfide (Capillary 3 i.d. 0.125 mm; DAPI-2 opening time 25 ms; q=0.35 for thiyl radical ions); (d) Ion/molecule reactions between isolated m/z 307 from panel (c) with dimethyl disulfide.

● S S S O + O + H CH SSCH H HOOC N COOH + H 3 3 HOOC N COOH + H N N H + ●SCH (47 Da) H NH O 3 NH O 2 m/z 307 2 m/z 354

Scheme 2.2

Multiple introductions of the reactant species within a single scan through DAPI-2 could be implemented using the method described previously.13d Higher reaction yield should be expected with longer DAPI opening time and/or higher number of neutral introduced; however, no significant difference in the reaction yield was observed for the different scan functions with different DAPI-2 opening times and various numbers of injections (Figure 2.8).

Figure 2.8 Thiyl radical reaction for different capillary inner diameters and opening times.

A new scan function was designed and implemented to isolate the thiyl radical ions m/z 307 (Figure 2.7c) after one reaction cycle and then allowed to react with the dimethyl disulfide introduced again from DAPI-2 (capillary 3 i.d. 0.125 mm; DAPI-2 opening time

25 ms). Surprisingly, no product ions were observed (Figure 2.7d). This indicates that a portion of thiyl radical ions had been converted into nonreactive species, presumably Cα- radical ions,17, 19 during the first reaction cycle. In fact, the energy barrier of thiyl radical

68 isomerization is relatively low (7.4 kcal/mol).17 It has been demonstrated that in-source fragmentation or in-trap CID can promote this process and lead to the loss of thiyl radical reactivity. In order to confirm this hypothesis, an ac excitation was used to activate the thiyl radicals immediately after they were produced and isolated; dimethyl disulfide was then introduced for reactions. The reaction yield decreased as the ac amplitude for excitation increased (Figure 2.9). Specifically, the reaction yield was only 15% with an ac excitation voltage of 0.07 V and the fragments of the thiyl radical ions, m/z 289 due to

+• 19 loss of water and m/z 232 ([b2-H] ) were also observed (Figure 2.9c).

Figure 2.9 MS spectra of thiyl radical reaction for activation with ac amplitude (a) 0.06 V; (b) 0.066 V; (c) 0.07 V; and (d) CID spectrum of thiyl radical ions.

These results suggest that more stable Cα-radical ions could be formed with energy deposition through an excitation, either by ac activation or gas flow. The side reactions might occur due to the gas flow with DAPI opening.

2.5 Conclusion

Use of simple instrument configuration of multiple DAPIs for gas-phase ion chemistry study was explored with a home built DAPI-RIT-DAPI system. The discontinuous atmospheric pressure interfaces enable introduction of multiple reactants with simple instrument configuration. The in-trap pressure varies during a scan, which also allows a reaction to be carried out at a high pressure while MS analysis of products at a much lower one. The gas dynamic effect on the internal energy deposition to the ions has been studied with thermometer ions. The ion/molecule reaction between glutathione thiyl radical and dimethyl disulfide was investigated to demonstrate the feasibilities of the

DAPI-RIT-DAPI system, where a radical immigration reaction occurred at the same time 69

of the dissociative addition reaction. With the simplicity of the configuration and the easiness in instrumentation modification, a variety of sources for ions and radicals, such as synchronized discharge ionization,25 low temperature plasma ionization,26 UV light irradiation,27 and pyrolysis nozzle,28 can also be coupled with this system in the future for investigation of the gas-phase reactions.

2.6 References

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11. (a) Pyatkivskyy, Y.; Ryzhov, V., Coupling of ion-molecule reactions with liquid chromatography on a quadrupole ion trap mass spectrometer. Rapid Commun. Mass Spectrom. 2008, 22 (8), 1288-1294; (b) Poad, B. L. J.; Pham, H. T.; Thomas, M. C.; Nealon, J. R.; Campbell, J. L.; Mitchell, T. W.; Blanksby, S. J., Ozone-Induced Dissociation on a Modified Tandem Linear Ion-Trap: Observations of Different Reactivity for Isomeric Lipids. J. Am. Soc. Mass Spectrom. 2010, 21 (12), 1989-1999. 12. (a) Badman, E. R.; Chrisman, P. A.; McLuckey, S. A., A quadrupole ion trap mass spectrometer with three independent ion sources for the study of gas-phase ion/ion reactions. Anal. Chem. 2002, 74 (24), 6237-6243; (b) Xia, Y.; McLuckey, S. A., Evolution of instrumentation for the study of gas-phase ion/ion chemistry via mass spectrometry. J. Am. Soc. Mass Spectrom. 2008, 19 (2), 173-189. 13. (a) Gao, L.; Sugiarto, A.; Harper, J. D.; Cooks, R. G.; Ouyang, Z., Design and characterization of a multisource hand-held tandem mass spectrometer. Anal. Chem. 2008, 80 (19), 7198-205; (b) Gao, L.; Cooks, R. G.; Ouyang, Z., Breaking the pumping speed barrier in mass spectrometry: Discontinuous atmospheric pressure interface. Anal. Chem. 2008, 80 (11), 4026-4032; (c) Huang, G.; Xu, W.; Visbal-Onufrak, M. A.; Ouyang, Z.; Cooks, R. G., Direct analysis of melamine in complex matrices using a handheld mass spectrometer. Analyst 2010, 135 (4), 705-711; (d) Gao, L.; Li, G.; Nie, Z.; Duncan, J.; Ouyang, Z.; Cooks, R. G., Characterization of a discontinuous atmospheric pressure interface. Multiple ion introduction pulses for improved performance. Int. J. Mass Spectrom. 2009, 283 (1-3), 30-34. 14. Chen, T.-C.; Xu, W.; Garimella, S.; Ouyang, Z., Study of the efficiency for ion transfer through bent capillaries. J. Mass Spectrom. 2012, 47 (11), 1466-1472. 15. Xu, W.; Charipar, N.; Kirleis, M. A.; Xia, Y.; Ouyang, Z., Study of Discontinuous Atmospheric Pressure Interfaces for Mass Spectrometry Instrumentation Development. Anal. Chem. 2010, 82 (15), 6584-6592. 16. Ouyang, Z.; Wu, G. X.; Song, Y. S.; Li, H. Y.; Plass, W. R.; Cooks, R. G., Rectilinear ion trap: Concepts, calculations, and analytical performance of a new mass

analyzer. Anal. Chem. 2004, 76 (16), 4595-4605. 71

17. Rauk, A.; Armstrong, D. A.; Berges, J., Glutathione radical: Intramolecular H abstraction by the thiyl radical. Can. J. Chem. 2001, 79 (4), 405-417. 18. Garimella, S.; Xu, W.; Ouyang, Z., Simulation of Transient Rarefied Gas Expansion for Discontinuous Atmospheric Pressure Interface (DAPI) Using Direct Simulation Monte Carlo (DSMC). American Society for Mass Spectrometry 60th Annual Conference Proceedings: Ion Manipulation, Analysis and Detection: New Developments 2012, ThOF (PM), 349. 19. Zhao, J.; Siu, K. W. M.; Hopkinson, A. C., Glutathione radical cation in the gas phase; generation, structure and fragmentation. Org. Biomol. Chem. 2011, 9 (21), 7384- 7392. 20. (a) Osburn, S.; Berden, G.; Oomens, J.; O'Hair, R. A. J.; Ryzhov, V., Structure and Reactivity of the N-Acetyl-Cysteine Radical Cation and Anion: Does Radical Migration Occur? J. Am. Soc. Mass Spectrom. 2011, 22 (10), 1794-1803; (b) Tan, L.; Xia, Y., Gas-Phase Reactivity of Peptide Thiyl (RS center dot),Perthiyl (RSS center dot), and Sulfinyl (RSO center dot) Radical Ions Formed from Atmospheric Pressure Ion/Radical Reactions. J. Am. Soc. Mass Spectrom. 2013, 24 (4), 534-542.

21. (a) Coon, J. J.; Syka, J. E. P.; Schwartz, J. C.; Shabanowitz, J.; Hunt, D. F., Anion dependence in the partitioning between proton and electron transfer in ion/ion reactions. Int. J. Mass Spectrom. 2004, 236 (1-3), 33-42; (b) Tang, X. J.; Thibault, P.; Boyd, R. K., Fragmentation Reactions of Multiply-Protonated Peptides and Implications for Sequencing by Tandem Mass-Spectrometry with Low-Energy Collision-Induced Dissociation. Anal. Chem. 1993, 65 (20), 2824-2834. 22. Tolmachev, A. V.; Vilkov, A. N.; Bogdanov, B.; Pasa-Tolic, L.; Masselon, C. D.; Smith, R. D., Collisional activation of ions in RF ion traps and ion guides: The effective ion temperature treatment. J. Am. Soc. Mass Spectrom. 2004, 15 (11), 1616-1628. 23. Huang, T. Y.; Emory, J. F.; O'Hair, R. A. J.; McLuckey, S. A., Electron-transfer reagent anion formation via electrospray ionization and collision-induced dissociation. Anal. Chem. 2006, 78 (21), 7387-7391. 24. (a) Gabelica, V.; De Pauw, E., Internal energy and fragmentation of ions produced in electrospray sources. Mass Spectrom. Rev. 2005, 24 (4), 566-587; (b) Nefliu, M.; Smith, J. N.; Venter, A.; Cooks, R. G., Internal energy distributions in desorption electrospray ionization (DESI). J. Am. Soc. Mass Spectrom. 2008, 19 (3), 420-427; (c) Wang, H.; Liu, J. J.; Cooks, R. G.; Ouyang, Z., Paper Spray for Direct Analysis of Complex Mixtures Using Mass Spectrometry. Angew. Chem.-Int. Edit. 2010, 49 (5), 877- 880. 25. Chen, T.-C.; Ouyang, Z., Synchronized Discharge Ionization for Analysis of Volatile Organic Compounds Using a Hand-Held Ion Trap Mass Spectrometer. Anal. Chem. 2013, 85 (3), 1767-1772. 26. Ma, X. X.; Love, C. B.; Zhang, X. R.; Xia, Y., Gas-Phase Fragmentation of [M + nH + OH](n center dot+) Ions Formed from Peptides Containing Intra-Molecular Disulfide Bonds. J. Am. Soc. Mass Spectrom. 2011, 22 (5), 922-930. 27. Stinson, C. A.; Xia, Y., Radical induced disulfide bond cleavage within peptides via ultraviolet irradiation of an electrospray plume. Analyst 2013, 138 (10), 2840-2846. 28. Zhang, X.; Friderichsen, A. V.; Nandi, S.; Ellison, G. B.; David, D. E.; McKinnon,

J. T.; Lindeman, T. G.; Dayton, D. C.; Nimlos, M. R., Intense, hyperthermal source of 72 organic radicals for matrix-isolation spectroscopy. Rev. Sci. Instrum. 2003, 74 (6), 3077- 3086.

CHAPTER 3. GAS-PHASE REACTIONS OF C3H2 WITH PROTONATED ALKYL AMINES: FORMATION OF A C-N COVALENT BOND

“The reaction between alkyl amines and C3H2 carbenes was a surprise in the study of bio-ion/radical reactions. However, science is sometimes not about how to follow the design and prediction, but discovering new things others never had.”

3.1 Introduction

Vinylidene carbenes (C3H2) are unsaturated divalent carbon species displaying two non-bonding electrons in a singlet or triplet configuration. There are three structural isomers of C3H2: cyclopropenylidene, propadienylidene, and propynylidene. Vinylidene carbenes overall are highly reactive and not stable in the condensed phase. However, they have been detected in hydrocarbon flames and are considered as the key intermediates for

1 soot formation; also, cyclopropenylidene (c-C3H2), a singlet carbene, is the most abundant cyclic hydrocarbons observed in the interstellar space.2 A significant amount of

3 effort has been made to synthesize relatively stable c-C3H2 derivatives. These derivatives have been widely utilized as ligands of transition metal catalysts in cross-coupling reactions.4

Given the important role of C3H2 species in the combustion chemistry, organic synthesis, and interstellar chemistry, it is highly desirable to establish a fundamental understanding of their physical and chemical properties. The relative energies,

geometries, and the isomerization among the three isomers of C3H2 have been well mapped with the experimental gas-phase studies and theoretical calculations.5

Cyclopropenylidene is the most stable among the three isomers. Driven toward forming a

+ stable aromatic system (c-C3H3 ) with 2 π-electrons, c-C3H2 has a gas-phase basicity (GB) of 916 kJ/mol from experimental measurements,6 which is significantly higher than the

7 GB of ammonia (819 kJ/mol). Investigation of c-C3H2 reactivity toward organic molecules so far has been rarely achieved by experimental methods because of its high instability in the condense phase.3 Instead, theoretical calculations have been used to study several types of reactions of c-C3H2 with organic molecules, including double bond addition,8 single bond insertion,9 and skeletal rearrangement.10 In this study, we experimentally studied gas-phase reactions of c-C3H2 with protonated alkyl amines for the first time.

3.2 Instrumentation

Our platform (Figure 2.1a) was constructed with a DAPI-RIT-DAPI configuration 74

(DAPI: discontinuous atmospheric pressure interface11; RIT: rectilinear ion trap12), which has been previously shown to provide high pressure and energetic gas flow for gas-phase ion/molecule reactions13. The ion trap served as both reactor and mass analyzer with a pyrolysis nozzle to generate radical species.

For general ion/radical reactions (MS scan function shown in Figure 2.1b), DAPI-1 was used to introduce alkyl ammoniums generated by nanoelectrospray (nanoESI) while

DAPI-2 connected to the pyrolysis nozzle was used to introduce vapor of carbene precursors (placed in a glass tube with a plastic Swagelok connected to capillary 3 of 0.25

75 mm i.d.). Ions of interest introduced through DAPI-1 were mass-selectively isolated and then interacting with carbenes from the dissociated precursors by pyrolysis from DAPI-2.

A glow discharge electron impact (GDEI)14 source was installed to emit electrons into the

RIT to ionize the neutral radicals or radical precursors that are subsequently characterized by MS analysis. This system represented a simple and intuitionistic approach to carry out and study gas-phase reactions and show the first experimental evidence of a gas-phase reaction between c-C3H2 and protonated ammoniums forming a C-N covalent bond.

Figure 3.1 (a) Schematic of the home-built platform for the study of reactive radical species and (b) the general MS scan function for ion/radical reactions (y-axis: voltage). DAPI: discontinuous atmospheric pres-sure interface; RIT: rectilinear ion trap; GDEI: glow discharge electron impact; rf: radio frequency; ac: alternative current.

A single-phase radio frequency (rf) was applied to trap ions at 920 kHz or 1490 kHz for mass range of m/z 80-600 or 20-200, respectively. The control electronics was directly adopted from a Griffin Minotaur 300 (FLIR Systems, Inc., Wilsonville, OR). The front endcap (close to DAPI-1) was a flat electrode with a center hole of 3 mm diameter for ion introduction, while a mesh electrode (18 x 18 mesh size, 0.015” wire diameter;

McMaster-Carr, Aurora, OH, USA) was used for the back endcap (close to DAPI-2). A dc of 10-30 V was applied on both end electrodes to create a dc trapping potential well along the z direction. Resonance ejection (q = 0.81), was implemented for MS scan with a dipolar ac signal at 324 and 524 kHz for rf frequency of 920 and 1490 kHz, respectively.

The frequency of rf 1490 kHz was used for m/z 20-180 to observe low mass ions while rf

920 kHz was used for m/z 100-600. The inductance and capacitance of the rf coil were changed to accomplish the two frequencies. The conversion dynode of the detector was grounded. The isolation of the trapped ions was achieved by forward/backward scan.

Collision-induced dissociation (CID) was implemented by dipolar excitation with a

single-frequency ac applied between the x electrodes of the RIT. In the mechanism study, 76

the low mass cutoff (LMCO) of CID was set to m/z 25 for rf 1490 kHz to trap low mass

+ CID fragments (eg. C3H3 , m/z 39).

The pyrolysis nozzle was first developed by Peter Chen to generate intense radical species,15 which has been an efficient means to produce and transfer intense radicals to gas phase16. Reactive species such as ethylny, viny, allyl, phenyl radical, and carbenes etc. were generated by resistance-heated SiC nozzle from corresponding precursors15, whose structures were then analyzed by infrared and electronic spectra. Flowing- afterglow selected-ion flow tube (SIFT)17 and Fourier transform ion cyclotron resonance

(FTICR)18 mass spectrometers were the two instruments reported to study ion/radical reactions by pyrolysis nozzle. Proton transfer, associative detachment, and radical recombination reactions were observed when using allyl radicals with hydroniums, ortho- benzynes with hydroxides, and allyl radicals with benzyl radical cations, respectively. In our setup, a silicon carbide (SiC) tube of 1 cm long, 1 mm i.d., 2 mm o.d. (Hexoloy SA,

Niagara Falls, NY) was employed as the resistance heater with an alumina tube

(8746K12, McMaster-Carr, Aurora, OH, USA) for insulation. The SiC and the Al2O3 tube were nested and cemented with ceramic adhesive (903HP, Cotronics, Brooklyn, NY) into the machinable Al2O3 flange (8484K78, McMaster-Carr, Aurora, OH, USA).

Stainless steel capillary 2 inside the pinch valve of DAPI-2 was directly inserted into the

Al2O3 tube without further sealing. Electrical contact was provided by graphite disks

(AXF-FQ Poco Graphite, Decatur, TX) and 0.127-mm tantalum-foil clips (Alfa Aesar,

Ward Hill, MA). Two light bulbs of 100 W and 200 W, parallel to each other and in series with the nozzle, were used as the ballast. Both nanoESI and radical precursors were

protected in Helium environment ca 760 Torr to prevent the heated SiC from oxidation. 77

The major differences for the pyrolysis nozzle in our platform compared to those in flowing-afterglow selected ion flow tube or Fourier transform ion cyclotron resonance is

1) the high pressure ca 10-100 mTorr in the vacuum chamber during precursor injection and 2) injection duration of 50 ms continuously instead of 200 µs with 20-40 Hz frequency. Therefore, the radicals produced through the pyrolysis nozzle were expected to have 103 times more collisions in our platform than that in high vacuum (10-5 Torr) and pyrolysis temperature was dropping during the opening. The high pressure and energetic gas flow are the two factors contributing to the formation of nonspecific C3H2 carbene

from various precursors. Among three structures of C3H2 carbene species, the most stable c-C3H2 is more likely to survive under the given environment.

Multiple triggers provided by the control electronics were used to synchronize the operations of DAPIs, GDEI and MS analysis. All the spectra presented are the average of

5-9 continuous scans.

3.3 Materials

Ionic reactants of alkyl amines and amino acids were purchased from Sigma

Chemical Co. (Sigma-Aldrich, St. Louis, MO, USA). Different concentrations (5-100 µM) of the samples were prepared with methanol for nanoESI with 0.2 % acetic acid added to increase the intensity of protonated species if necessary.

Radical precursors including allylchloride, 1,5-hexadiene, pentane, propargylchloride were purchased from Sigma Chemical Co. (Sigma-Aldrich, St. Louis,

MO, USA). Propargylbromide was purchased from TCI America (Portland, OR, USA),

and d12-pentane was purchased from Alfa Aesar (Ward Hill, MA, USA). The head-vapor 78

of radical precursors in Helium buffer was injected into the platform to generate radical species. 1,3-dibromopropyne was synthesized as C3H2 carbene precursors according to the previous papers.19 In short, 1,3-dibromopropyne was prepared in two steps: Propargyl

20 alcohol was converted to 1-bromopropyn-3-ol by triphenyl phosphite methiodide. PBr3 was used in benzene and then pyridine to replace the hydroxyl group with Br.19 The final product was distilled under reduced pressure with Argon.

3.4 Results and discussion

3.4.1 Carbene reaction with protonated alkylamines

We used 1,3-dibromopropyne (BrC≡CCH2Br) as a precursor for C3H2 formation by pyrolysis.19 Figure 3.2a shows a typical reaction spectrum of protonated n-heptyl amine

(m/z 116) and the pyrolysis products of 1,3-dibromopropyne. Three new peaks, m/z 114,

154 and 192, were observed after the reaction. The ions of m/z 154 and 192 correspond to a mass increase of 38 Da and 2 × 38 Da, respectively, from the precursor ion (m/z 116).

The peak at m/z 114 likely resulted from the dissociation of ion m/z 154 due to excitation by hot gas flow when opening DAPI-2 for introducing pyrolysis products.13 Collisional activation of ion m/z 154 (Figure 3.2b) yielded ion m/z 114 through a neutral loss of 40

Da (C3H4), which supported C3H2 adduct formation.

Figure 3.2 Carbene reaction spectra: (a) MS2 reaction of protonated n-heptylamine with heated 1,3-dibromopropyne; (b) MS3 CID of the reaction product; (c) MS2 reaction of 2 protonated n-heptylamine with heated d12-pentane; and (d) MS reaction of protonated n- heptylamine 18C6 complex with heated 1,3-dibromopropyne.

Besides 1,3-dibromopropyne, we found that analogous reaction products, viz. the mass increases of 38 Da, 2 × 38 Da, or even 3 × 38 Da to the protonated n-heptyl amine ions, were obtained when using other hydrocarbons as precursors for pyrolysis, including allylchloride, propargyl-bromide, 1,5-hexadiene, and pentane (Figure 3.3). The last reaction allowed us to use fully deuterated pentane (d12-pentane) as a pyrolysis precursor to confirm the chemical identity of the 38 Da adduct. The reaction spectrum is shown in

Figure 3.2c. Reaction products at m/z 156 and 196 correspond to mass increases of 40 Da

(C3D2) and 2 × 40 Da, respectively, from the amine ions (m/z 116), consistent with our conclusion that the addition indeed results from C3H2. All the reaction and their corresponding CID spectra of the test amines and pyrolysis precursors can be found in

Table 3.1 and Table 3.2.

Figure 3.3 MS2 reaction spectra of heptyl ammonium and heated (a) allylchloride, (b) propargylbromide, (c) 1,5-hexadiene, and (d) pentane.

We also investigated reactions of secondary, tertiary, and quaternary alkylammonium ions with C3H2. Similar reaction phenomena were observed for all the amine ions except for the quaternary alkylammonium ion, for which no reaction product was observed (Figure 3.4). This series of experiments indicated that an amine nitrogen and an ammonium proton were involved in the reaction. We further tested reactions of

C3H2 with the non-covalent complex of 18-crown-6 ether (18C6) and protonated n-heptyl amine. In such a complex, three strong hydrogen bonds are formed between the protonated primary amine and three oxygen atoms of 18C6, making neither the proton nor the nitrogen available for further reaction or binding. Under the same reaction condition previously used for the n-heptyl amine, the addition of C3H2 to the complex ion m/z 380 ([M+18C6+H]+) was not observed (Figure 3.2d).

Figure 3.4 MS2 reaction spectra of protonated (a) dibutylamine, (b) tributylamine, and (c) cetyltrimethyl ammonium with heated propargylbromide, and MS3 CID spectra of the reaction products for (d) dibutylamine and (e) tributylamine.

The studies reported above suggest that protonation is an important factor for observing the C3H2 addition reaction. In order to gain further evidence, we compared the

C3H2 reactions with proline methyl ester ions in protonated vs. sodium adduct forms. The most favorable protonation site of proline methyl ester is at the pyrrolidine nitrogen, while for the sodium adduct, the sodium ion is chelated by the carbonyl oxygen and the amine nitrogen atoms.21 The reaction course for these two ions was drastically different.

While an efficient C3H2 addition was observed for the protonated ions, the reaction yield was extremely low for the sodium adduct ions (Figure 3.5). This observation is consistent with the results for the alkyl amines, for which only the protonated amines undergo efficient C3H2 additions.

Figure 3.5 MS2 reaction spectra of (a) protonated and (b) sodiated proline methyl ester with heated allylchloride, and their corresponding product MS3 CID spectra (c) and (d), 82 respectively.

C3H2 has three known isomeric structures, viz. cyclopropenylidene (c-C3H2), propadienylidene, and propynylidene which is a 3A” triplet in the ground electronic state.5d To explain the course of the ammonium ion addition, it is important to identify which structure(s) are involved in the observed reactions. When GDEI was used to identify the pyrolysis product(s), the different MS patterns with heating on and off (Table

3.3) prove that new species were generated through pyrolysis. The GDEI data is not conclusive but indicative for the identity of C3H2 since extensive ion/molecule reactions

83 would occur after the initial electron impact at the high pressure (10 mTorr). The fact that a variety of hydrocarbons could all produce C3H2 suggests that the C3H2 formation might not be closely tied to the pyrolysis chemistry; instead, it is most likely determined by the energetics and reactions after the pyrolysis. Note that the ion trap pressure was relatively high (10-100 mTorr) when the pyrolysis valve was open, and the free sonic expansion was shorter than 2 cm, while the distance between the nozzle and the center of the RIT is

5 cm. The first generation pyrolysis products can undergo extensive and energetic

13, 22 collisions as they are entering the ion trap, be converted to C3H2, which subsequently reacts with the trapped ions. Since the reaction chemistry involves the protons on the ammonium ion, we suspect that c-C3H2, which has a relatively high GB, 916 kJ/mol at

1 1 298 K for the A1 state, as compared to 860 kJ/mol for ( A1) propadienylidene, and 687 kJ/mol for the 3A” state of propynylidene, might be the dominant species formed by pyrolysis. In order to clarify the reaction mechanism, further evidence was sought from theoretical calculations and additional experiments.

3.4.2 Theoretical calculation of the reaction mechanism

Since C3H2 has three identified isomeric structures, i.e., cyclopropenylidene (c-

C3H2), propadienylidene (l-C3H2), and propynylidene (l-C3H2), it is of importance to know which structure(s) are involved in the observed reactions. We tested a variety of hydrocarbons including allylchloride, 1,5-hexadiene, propargyl-bromide, and pentane as

C3H2 precursors and observed the same reaction phenomenon as shown in Figure 3.2a, where 1,3-dibromopropyne was used as the C3H2 precursor and reacted with protonated alkyl amine. These results suggest that the C3H2 formation is not closely tied to the

84 pyrolysis chemistry or process, instead mostly determined by the energetics and reactions after pyrolysis. Note that the ion trap pressure is relatively high (10-100 mTorr) upon the opening of the pyrolysis valve of the current instrument design, which gives only less than 2 cm free sonic expansion of the radical species after the initially formed from pyrolysis in comparison with 4 cm between the nozzle and the center of the RIT. These radical species undergo extensive and energetic collisions as they enter the trap13, 22 and are further converted into other products. We suspect that c-C3H2 might be the dominant species formed from such process since c-C3H2 has lower energy by about 42–59 and 59–

92 kJ/mol than that of the propadienylidene and propynylidene.1, 5a, 23 This hypothesis is also supported by the reaction chemistry which is mainly driven by proton. Indeed, c-

C3H2 has a significant GB of 916 kJ/mol as mentioned above. A reaction mechanism of proton-bound dimer formation between c-C3H2 and protonated alkyl amine as the first step followed by C-N covalent bond formation was proposed to explain the observed reaction phenomenon in Figure 3.2a. This mechanism was further investigated and

evaluated by theoretical calculations and experimental results. 84

+ Figure 3.6 Calculated PES of (a) CH3NH3 /c-C3H2 reaction to form a C-N covalent bond from Brauman double-well complexes (numbers: CCSD(T)/aug-cc-pVTZ, kJ/mol) and (b) the two Brauman intermediates as a function of the distance of N-H---C, blue square: CCSD(T)/aug-cc-pVTZ single point energies on M06-2X/6-311+G(2d,p) optimized geometries; open circle: M06-2X/6-311+G(2d,p) optimized; filled circle: B3LYP/6- 311+G(2d,p) optimized.

+ The reaction between c-C3H2 and protonated methyl amine (CH3NH3 ) was used as 85

a simplified model for theoretical calculations at the CCSD(T)/aug-cc-pVTZ//M06-2X/6-

311+G(2d,p) level of theory and including zero point vibrational energies. The key structures involved in the reaction together with their 0 K enthalpies (relative to the enthalpy of the reactants) are illustrated in Figure 3.6a. The first step of the reaction is the

+ formation of a proton-bound dimer between c-C3H2 and CH3NH3 . This step is exothermic, producing two Brauman intermediates, Complex 1 (-127 kJ/mol) and

Complex 2 (-121 kJ/mol) that were found as the structures with the lowest energies

(Figure 3.6b). Complex 2 has a structure with a bonding distance between the proton and

the carbon of the c-C3H2 (N-H---C = 1.16 Å), as compared to Complex 1 (N-H---C =

1.72 Å), which indicates that the proton moved from the amine nitrogen to the c-C3H2 moiety. This shift places a partial positive charge on the C1 of c-C3H2 and facilities a subsequent nucleophilic attack by the amine nitrogen, leading to the formation of a C-N bond in product A (-216 kJ/mol) through a low energy barrier. Product A might quickly go through rearrangements, such as ring opening and hydrogen atom migration (TS1 in

Figure 3.6a), to form a more stable product B (-372 kJ/mol). Product B is shown with a cis-syn geometry that can isomerize to a slightly more stable trans-isomer. In either isomer, two of the original amine hydrogens are transferred to the C3H4 moiety. It is worth noting that Complex 1 has the proton more tightly bound to the amine nitrogen and, as a result, the N-C covalent bond formation has a high energy barrier (Figure 3.7).

Figure 3.7 Calculated PES of N--H--C angle graph of Complex 1, 2 and product A

3.4.3 Experimental evidences of the reaction mechanism

The mechanism proposed in Figure 3.6 shows that the formation of a proton-bound dimer between c-C3H2 and protonated alkyl amine is the key step for the overall reaction.

The proton-bound dimer can stay as it is (Complex 1) or undergo further rearrangement

87 to form a covalent product. Although the proton-bound dimer and the covalent product has the same m/z (indicated as [C]), they should be distinguished via unique unimolecular dissociation channels upon collisional activation. For instance, loss of 40 Da (m/z [C-40]) is the signature fragmentation channel from the covalent product as shown in Figure 3.2b.

CID of proton-bound dimer, however, leads to the separation of c-C3H2 and alkyl amine.

+ Proton transfer product, C3H3 (m/z 39), and protonated amines (m/z [C-38]) should be the main products, and their relative abundances are dependent on the gas-phase basicities of the corresponding neutrals. A breakdown curve of protonated butylamine/carbene reaction as an example is illustrated in Figure 3.8 to show the relatively abundance of these three CID products together with the precursor complex as a function of the resonance activation ac amplitude. The yields of three products are relatively stable throughout the range of the activation energies, which implies that the three products have similar energy barriers for dissociation.

Figure 3.8 The breakdown curve of protonated butylamine/carbene reaction by collisional activation: relative intensity of parent complex ([C], blue circle), covalent product ([C- 40], red diamond), proton-transfer product (m/z 39, green triangle), and direct dissociation ([C-38], green square) as a function of activation energy.

The relative intensity of [C-40] among all fragment ions can be used to indicate the yield of the covalent product (% Covalent Complex). If the mechanism proposed in

Figure 3.6 truly reflects the nature of the reaction, % Covalent Complex could be varied by changing the gas-phase basicity of the amines. Using the calculated GB of c-C3H2 920 kJ/mol as a bench mark (CCSD(T)/aug-cc-pVTZ single-point energies on CCSD/6-

311+G(2d,p) optimized geometries), we investigated reactions of c-C3H2 produced by pyrolysis of allylchloride with a series of protonated alkyl amines and guanidine, with their GBs ranging from 884 to 949 kJ/mol.7 The complexes ([C]) formed by these reactions were subjected to CID to achieve 80-90% of dissociation.

The CID spectra of complexes formed by c-C3H2 addition to protonated n-butyl amine, ethylmethyl amine, di-n-propylamine, and guanidine are shown in Figure 3.9a-d.

They represent cases that amines have GBs lower, comparable, higher, and significant higher than that of c-C3H2, respectively. CID of the butylamine-C3H2 complex (m/z 112) generates [C-40] (m/z 72) and m/z 39 at about equal relative intensity while the intensity

of [C-38] (m/z 74) is very low (Figure 3.9a). This is because the GB of n-butylamine 88

(887 kJ/mol) is smaller than that of c-C3H2 which is therefore more favorably protonated.

Based on the data shown in Figure 3.9a, the yield of the covalent complex is about 52%.

The CID spectrum of the ethylmethylamine-C3H2 complex (Figure 3.9b) shows the loss of 40 Da (m/z 58) as the main fragmentation, leading to 68 % Covalent Complex. Given the similar GBs of ethylmethylamine (909 kJ/mol) and c-C3H2, the ion intensities of

+ C3H3 (m/z 39) and protonated amine (m/z 60) are comparable. When the GB is further increased, as in the case of di-n-propylamine, (GB= 929 kJ/mol), only two fragment channels are observed, [C-40] and [C-38], while m/z 39 intensity diminishes as a result of

a lower GB of c-C3H2 relative to the amine. The % Covalent Complex is 49%. Guanidine has the highest GB (949 kJ/mol)24 among all the species tested. CID of the complex shows a loss of neutral carbene ([C-38], m/z 60) as the only fragmentation pathway.

Figure 3.9e summarizes the correlation between the % Covalent Complex and the

GB values of various alkyl amines and guanidine. The corresponding CID spectra are shown in Table 3.2. The highest yield (82%) for the formation of a covalent product is obtained for cyclohexylamine, which has a GB value slightly lower than c-C3H2. The yield decreases as the GB difference becomes larger. The fact that covalent complex formation is most favored with a minimal GB difference25 between the amines and c-

C3H2 strongly supports the formation of the proton-bound dimer is the critical step, as pointed out by the theoretical calculations (Figure 3). Furthermore, it also supports that c-

1 C3H2 instead of other two isomers is mainly responsible for the reaction. The A1 ground state of propadienylidene is both substantially less stable (+53 kJ/mol) and less basic (GB

3 = 860 kJ/mol) than c-C3H2. Propynylidene exists as a high-energy triplet state ( A”, +47

+ kJ/mol) of low basicity (GB = 687 kJ/mol) when forming a linear triplet C3H3 cation. 89

Figure 3.9 Selected MS3 reaction product CID spectra of (a) butylamine, (b) ethylmethylamine, (c) dipropylamine, and (d) guanidine with c-C3H2 and (e) %Covalent of protonated alkyl amines/c-C3H2 reactions as a function of GBs of the corresponding alkyl amines. The %Covalent is calculated as I[c-40]/(I[39]+I[c-40]+I[c-38]), where I[39], I[c-40], and I[c-38] correspond to the intensity of m/z 39 as the proton-transfer product, neutral loss

of 40 Da as the covalent product, and neutral loss of 38 Da as the direct-dissociation 90 product, respectively, in the MS3 CID spectra based on 80-90% parent ions fragmented. Error bars were based on 5 duplicates of an average of 3 spectra.

We also formed amine-C3H2 proton-bound dimer from reactions of neutral amines

+ (i.e. heptylamine) and C3H3 ions. Ethyl, propyl, butyl, hexyl and heptyl amines were

+ investigated for ion/molecule reaction with c-C3H3 ions. Table 1.1 summarized the ion/molecule reaction spectra and their corresponding MS3 CID spectra. In comparison with corresponding ion/carbene reactions, complexes of exactly the same m/z values were observed for the reactions with neutral amines. Collisional activation of the heptylamine-

+ C3H3 complex (Figure 3.10) produced a very similar fragmentation pattern to that of the

C3H2-protonted heptyl amine product shown in Figure 3.2b. The above result also supports the reaction mechanism with the proton-bound dimer intermediate suggested for the ion-carbene reactions.

3 + Figure 3.10 MS reaction product CID spectrum of C3H3 and neutral n-heptyl amine

3.5 Conclusion

In summary, the home-developed DAPI-ion trap-pyrolysis MS platform facilitated studies of reactions of c-C3H2 with protonated alkyl amines in the gas phase. Addition of

C3H2 to the protonated amine was observed as the only reaction channel. Mechanistic

91 studies demonstrate that non-covalent and covalent (via C-N formation) complexes can be produced and they both stem from the formation of proton-bound dimers as the key step. The covalent product formation is in favor of alkyl amines with similar GB values as that of the c-C3H2. In the interstellar carbon chemistry, it has been long believed that the major pathway for the formation of c-C3H2 comes from electron attachment to c-

+ 26 C3H3 , while the major pathway for the destruction of c-C3H2 goes through the

+ protonation of c-C3H2 to form c-C3H3 , which makes c-C3H2 a “dead end” in this carbon cycle.26-27 This study therefore provides a new perspective to other possible reaction

pathways of c-C3H2 in the interstellar medium. In the presence of protonated amines or even ammonium, c-C3H2 can form covalent adducts to seed the formation of larger organic molecules/ions consisting of C, N, and H atoms.

3.6 Appendix

Table 3.1 summarizes MS2 reaction spectra of all alkyl ammoniums investigated with C3H2 generated by the pyrolysis of various organic precursors. The number of +38

Da adducts was corresponding to the number of protons on the charged amine site. The nonspecific generation of C3H2 carbenes was observed from precursors with three or more hydrocarbons. Therefore, cyclopropenylidene was most-likely the carbene species produced during pyrolysis because it is more stable compared to the other two C3H2 carbenes. The series of adducts could be both covalent products and simple complexes, thus CIDs were then used for structure elucidation on the selected carbene reaction products.

Table 3.1 Ammonium reactions with various radical precursors Radical Reactant MS Spectrum No. Precursor

BrC≡CCH2Br 1,3- 1 Dibromopropyne NH2 Heptylamine [M+H]+ m/z 116 Cl 2 Allylchloride

Br 3 Allylbromide

I 4 Allyliodide

Br 5 Propargylbromide

1, 5-Hexadiene

7 Pentane

D2 D2 C C D3C C CD3 8 D2 Pentane-D12

NH2 Heptylamine 18-Crown-6 9 [M+18C6+H]+ Propargylbromide m/z 380

Heptylamine 12-Crown-4 10 [M+18C6+H]+ Propargylbromide m/z 292

D D N D Heptylamine H/D 11 [M-2H+3D]+ m/z 119 1, 5-Hexadiene

Methylamine Br 12 [M+H]+ Propargylbromide m/z 32

Ethylamine 13 + [M+H] m/z 46 1, 5-Hexadiene

Cl Propylamine 14 [M+H]+ Allylchloride m/z 60

Butylamine 15 [M+H]+ Allylchloride m/z 74

16 Isobutylamine Allylchloride [M+H]+ m/z 74

Hexylamine 17 [M+H]+ Allylchloride m/z 102

18 Cyclohexylamine Allylchloride [M+H]+ m/z 100

Cl Ethylmethylamine 19 [M+H]+ Allylchloride m/z 60

Diethylamine 20 [M+H]+ Allylchloride m/z 74

Dipropylamine 21 [M+H]+ Allylchloride m/z 102

22 Allylchloride

H N Br Dibutylamine 23 [M+H]+ Propargylbromide m/z 130

1, 5-Hexadiene

D D N Dibutylamine H/D 25 [M-H+2D]+ Propargylbromide m/z 132

26 Diisopropylamine Allylchloride [M+H]+ m/z 102

Cl 27 Guanidine Allylchloride [M+H]+ m/z 60

28 Allylchloride

Br N 29 Tributylamine Propargylbromide [M+H]+ m/z 186

1, 5-Hexadiene

N D 31 Tributylamine H/D Propargylbromide [M+D]+ m/z 187

CH 32 3 Propargylbromide H3C(H2C)15 N CH3 CH3 Cetyltrimethyl ammonium M+ m/z 284 33

1, 5-Hexadiene

Ion-trap CID for the carbene adducts was performed (shown in Table 3.2) to illustrate three reaction channels discussed in 3.4.3. For the data points in Figure 3.9e of

%Covalent complex vs. GB curve, a LMCO of m/z 25 was used for all MS2 reactions and

MS3 CIDs with the rf frequency of 1490 kHz. The rf frequency of 920 kHz was used to trap ions larger than m/z 100 and to record the rest reaction and CID spectra. The final stable product shown in Figure 3.6 had two of the original hydrogens of the carbene side,

3 so that deuterium scrambling was expected in MS CID spectra when using d12-pentane as the carbene precursor (CID No.44) and H/D exchanged heptyl ammonium (CID

No.45).

Table 3.2 MS3-MS5 CID spectra of selected reaction products Selected CID Reaction No. MS Spectrum CID No. precursor m/z

MS3 1 34 116-154

MS3 2 35 116-154

MS3 2 36 116-192

MS4 2-36 37 116-192-154

MS3 2 38 116-230

MS4 2-38 39 116-230-192

MS5 2-38-39 116-230-192- 40 154

MS3 5 41 116-154

MS3 6 42 116-154

MS3 7 43 116-154

MS3 8 44 116-156

MS3 11 45 116-157

MS3 13 46 46-84

MS3 14 47 60-98

MS3 15 48 74-112

MS3 16 49 74-112

MS3 17 50 102-140

MS3 18 51 100-138

MS3 19 52 60-98

MS3 20 53 74-112

MS3 21 54 102-140

100

MS3 22 55 130-168

MS3 22 56 130-206

MS4 22-56 57 130-206-168

MS3 26 58 102-140

MS3 27 59 60-98

100

MS3 28 60 186-224

The internal ion source GDEI was designed to show the MS characterization with pyrolysis on or off in Table 3.3. However, the radical precursors are usually easy to

+ 28 generate reactive ion species (eg. C3H3 ) by electron impact ionization. Based on the pressure of 10-100 mTorr after DAPI opening, further reactions would occur between ions and neutral precursors (also the impurity in the precursor) at the number density of

101

3.3-33x1014 cm-3. Therefore, the GDEI spectra could not directly reflect the radical species generated by pyrolysis. Since the reactions between alkyl ammoniums and radicals from various precursors to form +38 covalent adducts have strongly supported the generation of C3H2 carbene species by the pyrolysis nozzle, the purpose of GDEI characterization was just to observe the pattern changing when the pyrolysis nozzle was turned on or off. The changing of GDEI pattern between room temperature and heated precursors indicated the formation of new species by pyrolysis for all the precursors, which was considered an indirectly evidence of C3H2 carbene reaction.

Table 3.3 GDEI spectra of radical precursors Precursor Pyrolysis MS Spectrum

Off

BrC≡CCH2Br 1,3-Dibromopropyne

101

Off

Allylchloride

102

Off

Allylbromide

Off

Allyliodide

Off

Propargylbromide 102

Off

1, 5-Hexadiene On

103

Off

Pentane

+ C3H3 ion/molecule reactions have been previously studied with multiple reaction channels observed such as interactions with unsaturated hydrocarbons,28-29 proton transfer and complexes.30 However, no CID results were reported to study the final structure of the products, probably due to the instrumentational setup. In this section, we used

+ propargylchloride to generate a majority of c-C3H3 ions by GDEI as comparison to the proposed c-C3H2 carbene reactions. It was previously shown that propargylchloride

+ produced 85-90% of non-reactive cyclic C3H3 ions by electron impact or chemical ionization.28 No effort was made to further separate linear (reactive) and cyclic (non-

103

+ + reactive) C3H3 species. After isolating C3H3 m/z 39 ions with rf 1490 kHz at a LMCO of m/z 10, neutral amines were injected through DAPI-1 with an additional capillary. The reaction took place at LMCO of m/z 25, which allowed the trapping of m/z 39-180. CIDs were performed at LMCO of m/z 25 as well. The MS3 CID spectra of ion/molecule reactions in Table 1.1 were very similar to those of ion/carbene reactions in Table 3.2, which proved they shared common reaction mechanism by forming a proton-bound dimer.

104

+ Table 3.4 Ion/molecule reactions of c-C3H3 and neutral amines Reactants MS type MS Spectrum

MS2 39-

+ c-C3H3 + Ethylamine

MS3 CID 39-84-

MS2 39-

+ c-C3H3 + Prpylamine

MS3 CID 39-98-

MS2 39-

+ 104 c-C3H3 +

Butylamine

MS3 CID 39-112-

MS2 39-

+ c-C3H3 + Hexylamine MS3 CID 39-140-

105

MS2 39-

+ c-C3H3 + Heptylamine MS3 CID 39-154-

105

106

3.7 References

1. Taatjes, C. A.; Klippenstein, S. J.; Hansen, N.; Miller, J. A.; Cool, T. A.; Wang, J.; Law, M. E.; Westmoreland, P. R., Synchrotron photoionization measurements of combustion intermediates: Photoionization efficiency and identification of C3H2 isomers. Phys. Chem. Chem. Phys. 2005, 7 (5), 806-813. 2. (a) Thaddeus, P.; Vrtilek, J. M.; Gottlieb, C. A., LABORATORY AND ASTRONOMICAL IDENTIFICATION OF CYCLOPROPENYLIDENE, C3H2. Astrophys. J. 1985, 299 (1), L63-L66; (b) Madden, S. C.; Irvine, W. M.; Matthews, H. E.; Friberg, P.; Swade, D. A., A SURVEY OF CYCLOPROPENYLIDENE (C3H2) IN GALACTIC SOURCES. Astron. J. 1989, 97 (5), 1403-1422. 3. Lavallo, V.; Canac, Y.; Donnadieu, B.; Schoeller, W. W.; Bertrand, G., Cyclopropenylidenes: From interstellar space to an isolated derivative in the laboratory. Science 2006, 312 (5774), 722-724. 4. (a) Wass, D. F.; Haddow, M. F.; Hey, T. W.; Orpen, A. G.; Russell, C. A.; Wingad, R. L.; Green, M., Cyclopropenylidene carbene ligands in palladium C-C coupling catalysis. Chem. Commun. 2007, (26), 2704-2706; (b) Kuchenbeiser, G.; Donnadieu, B.; Bertrand, G., Stable bis(diisopropylamino)cyclopropenylidene (BAC) as ligand for transition metal complexes. J. Organomet. Chem. 2008, 693 (5), 899-904; (c) Wilde, M. M. D.; Gravel, M., Bis(amino)cyclopropenylidenes as Organocatalysts for Acyl Anion and Extended Umpolung Reactions. Angew. Chem., Int. Ed. 2013, 52 (48), 12651-12654. 5. (a) Seburg, R. A.; Patterson, E. V.; Stanton, J. F.; McMahon, R. J., Structures, automerizations, and isomerizations of C3H2 isomers. J. Am. Chem. Soc. 1997, 119 (25), 5847-5856; (b) Mebel, A. M.; Jackson, W. M.; Chang, A. H. H.; Lin, S. H., Photodissociation dynamics of propyne and allene: A view from ab initio calculations of the C3Hn (n=1-4) species and the isomerization mechanism for C3H2. J. Am. Chem. Soc. 1998, 120 (23), 5751-5763; (c) Vazquez, J.; Harding, M. E.; Gauss, J.; Stanton, J. F., High-Accuracy Extrapolated ab Initio Thermochemistry of the Propargyl Radical and the 106

Singlet C3H2 Carbenes. J. Phys. Chem. A 2009, 113 (45), 12447-12453; (d) Wu, Q.; Cheng, Q.; Yamaguchi, Y.; Li, Q.; Schaefer, H. F., III, Triplet states of cyclopropenylidene and its isomers. J. Chem. Phys. 2010, 132 (4). 6. Chyall, L. J.; Squires, R. R., DETERMINATION OF THE PROTON AFFINITY AND ABSOLUTE HEAT OF FORMATION OF CYCLOPROPENYLIDENE. Int. J. Mass Spectrom. Ion Processes 1995, 149, 257-266. 7. Hunter, E. P. L.; Lias, S. G., Evaluated gas phase basicities and proton affinities of molecules: An update. J. Phys. Chem. Ref. Data 1998, 27 (3), 413-656. 8. (a) Tan, X. J.; Li, Z.; Sun, Q.; Li, P.; Wang, W. H., Theoretical Study on the Mechanism of the Addition Reaction between Cyclopropenylidene and Formaldehyde. Bull. Korean Chem. Soc. 2012, 33 (6), 1934-1938; (b) Tan, X. J.; Li, Z.; Sun, Q.; Li, P.; Wang, W. H.; Wang, G. R., A Theoretical Study on the Mechanism of the Addition Reaction between Cyclopropenylidene and Ethylene. J. Chil. Chem. Soc. 2012, 57 (2), 1174-1177; (c) Li, Q. L.; Sun, Q.; Gu, J. S.; Tan, X. J., A computational study of the addition reaction of cyclopropenylidene with methyleneimine. Russ. J. Phys. Chem. A 2013, 87 (5), 806-812.

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9. (a) Jing, Y.; Liu, H.; Wang, H. L.; Yu, Y.; Tan, X. J.; Chen, Y. G.; Zhang, Y. L.; Gu, J. S., Quantum Mechanical Study of the Insertion Reaction between Cyclopropenylidene with R-H (R=F, Oh, Nh2, Ch3). J. Chil. Chem. Soc. 2013, 58 (4), 2218-2221; (b) Tan, X. J.; Li, Z.; Sun, Q.; Li, P.; Wang, W. H.; Wang, M. Y.; Chen, Y. G., Theoretical study on the reaction mechanisms between propadienylidene and R-H (R=F, OH, NH2, CH3): an alternative approach to the formation of alkyne. Struct. Chem. 2013, 24 (1), 33-38. 10. (a) Tan, X. J.; Wang, W. H.; Jing, Y.; Wang, F.; Li, P., Theoretical study on the reaction mechanism of cyclopropenylidene with azacyclopropane: ring expansion process. Monatsh. Chem. 2014, 145 (7), 1109-1115; (b) Tan, X. J.; Wang, W. H.; Sun, Q.; Jing, Y.; Li, P., Theoretical study of the ring expansion reaction mechanism of cyclopropenylidene with azetidine. J. Mol. Model. 2014, 20 (3). 11. Gao, L.; Cooks, R. G.; Ouyang, Z., Breaking the pumping speed barrier in mass spectrometry: Discontinuous atmospheric pressure interface. Anal. Chem. 2008, 80 (11), 4026-4032. 12. Ouyang, Z.; Wu, G. X.; Song, Y. S.; Li, H. Y.; Plass, W. R.; Cooks, R. G., Rectilinear ion trap: Concepts, calculations, and analytical performance of a new mass analyzer. Anal. Chem. 2004, 76 (16), 4595-4605. 13. Lin, Z.; Tan, L.; Garimella, S.; Li, L.; Chen, T.-C.; Xu, W.; Xia, Y.; Ouyang, Z., Characterization of a DAPI-RIT-DAPI System for Gas-Phase Ion/Molecule and Ion/Ion Reactions. J. Am. Soc. Mass Spectrom. 2014, 25 (1), 48-56. 14. Gao, L.; Song, Q.; Noll, R. J.; Duncan, J.; Cooks, R. G.; Zheng, O., Glow discharge electron impact ionization source for miniature mass spectrometers. J. Mass Spectrom. 2007, 42 (5), 675-680. 15. Kohn, D. W.; Clauberg, H.; Chen, P., FLASH PYROLYSIS NOZZLE FOR GENERATION OF RADICALS IN A SUPERSONIC JET EXPANSION. Rev. Sci. Instrum. 1992, 63 (8), 4003-4005. 16. Zhang, X.; Friderichsen, A. V.; Nandi, S.; Ellison, G. B.; David, D. E.; McKinnon, J. T.; Lindeman, T. G.; Dayton, D. C.; Nimlos, M. R., Intense, hyperthermal source of 107 organic radicals for matrix-isolation spectroscopy. Rev. Sci. Instrum. 2003, 74 (6), 3077- 3086. 17. Zhang, X.; Kato, S.; Bierbaum, V. M.; Nimlos, M. R.; Ellison, G. B., Use of a flowing afterglow SIFT apparatus to study the reactions of ions with organic radicals. J. Phys. Chem. A 2004, 108 (45), 9733-9741. 18. Russell, A. L.; Rohrs, H. W.; Read, D.; Giblin, D. E.; Gaspar, P. P.; Gross, M. L., Radical cation/radical reactions: A Fourier transform ion cyclotron resonance study of allyl radical reacting with aromatic radical cations. Int. J. Mass Spectrom. 2009, 287 (1-3), 8-15. 19. Clauberg, H.; Minsek, D. W.; Chen, P., Mass and Photoelectron-Spectroscopy of C3h2 - Delta-Hf of Singlet Carbenes Deviate from Additivity by Their Singlet Triplet Gaps. J. Am. Chem. Soc. 1992, 114 (1), 99-107. 20. Greaves, P. M.; Kalli, M.; Landor, P. D.; Landor, S. R., Allenes .21. Preparation of 1,1-Dialkyl-3-Iodoallenes and 1,1-Dihalogenoallenes. Journal of the Chemical Society C-Organic 1971, (4), 667-&.

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21. Talley, J. M.; Cerda, B. A.; Ohanessian, G.; Wesdemiotis, C., Alkali metal ion binding to amino acids versus their methyl esters: Affinity trends and structural changes in the gas phase. Chem.-Eur. J. 2002, 8 (6), 1377-1388. 22. Garimella, S.; Zhou, X.; Ouyang, Z., Simulation of Rarefied Gas Flows in Atmospheric Pressure Interfaces for Mass Spectrometry Systems. J. Am. Soc. Mass Spectrom. 2013, 24 (12), 1890-1899. 23. Maier, G.; Preiss, T.; Reisenauer, H. P.; Hess, B. A.; Schaad, L. J., Small Rings .82. Chlorinated Cyclopropenylidenes, Vinylidenecarbenes, and Propargylenes - Identification by Matrix-Isolation Spectroscopy. J. Am. Chem. Soc. 1994, 116 (5), 2014- 2018. 24. Yao, C.; Syrstad, E. A.; Tureček, F., Electron transfer to protonated beta-alanine N-methylamide in the gas phase: An experimental and computational study of dissociation energetics and mechanisms. J. Phys. Chem. A 2007, 111 (20), 4167-4180. 25. (a) Davidson, W. R.; Sunner, J.; Kebarle, P., HYDROGEN-BONDING OF WATER TO ONIUM IONS - HYDRATION OF SUBSTITUTED PYRIDINIUM IONS AND RELATED SYSTEMS. J. Am. Chem. Soc. 1979, 101 (7), 1675-1680; (b) Larson, J. W.; McMahon, T. B., FORMATION, THERMOCHEMISTRY, AND RELATIVE STABILITIES OF PROTON-BOUND DIMERS OF OXYGEN N-DONOR BASES FROM ION-CYCLOTRON RESONANCE SOLVENT-EXCHANGE EQUILIBRIA MEASUREMENTS. J. Am. Chem. Soc. 1982, 104 (23), 6255-6261; (c) Bian, L., Proton donor is more important than proton acceptor in hydrogen bond formation: A universal equation for calculation of hydrogen bond strength. J. Phys. Chem. A 2003, 107 (51), 11517-11524. 26. Maluendes, S. A.; McLean, A. D.; Herbst, E., Calculations Concerning Interstellar Isomeric Abundance Ratios for C3h and C3h2. Astrophys. J. 1993, 417 (1), 181-186. 27. Millar, T. J.; Farquhar, P. R. A.; Willacy, K., The UMIST database for astrochemistry 1995. Astron. Astrophys. Suppl. Ser. 1997, 121 (1), 139-185. 28. Ozturk, F.; Baykut, G.; Moini, M.; Eyler, J. R., REACTIONS OF C3H3+ WITH 108

ACETYLENE AND DIACETYLENE IN THE GAS-PHASE. J. Phys. Chem. 1987, 91 (16), 4360-4364. 29. Smyth, K. C.; Lias, S. G.; Ausloos, P., THE ION-MOLECULE CHEMISTRY OF C3H3+ AND THE IMPLICATIONS FOR SOOT FORMATION. Combust. Sci. Technol. 1982, 28 (3-4), 147-154. 30. Prodnuk, S. D.; Gronert, S.; Bierbaum, V. M.; Depuy, C. H., GAS-PHASE REACTIONS OF C3HN+ IONS. Org. Mass Spectrom. 1992, 27 (4), 416-422.

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CHAPTER 4. GAS-PHASE C-C3H2 CARBENE REACTIONS WITH BIOMOLECULAR IONS

4.1 Introduction

It has been discussed in the previous chapter that c-C3H2 can be generated by nonspecific pyrolysis under the conditions in DAPI-RIT-DAPI mass spectrometer with 10-2

Torr pyrolysis pressure and energetics gas flows. The reactions between protonated alkyl amines and c-C3H2 have been studied and covalent products have been observed to form through a proton-bound dimer mechanism with further rearrangements.

Biomolecules such as nucleobases, nucleosides, amino acids, peptides, and proteins are more complicated than alkyl amines, which also contain amine functional groups. The protonated biomolecules are expected to be modified by c-C3H2 carbene through proton-

bound dimer, possibly to form covalent reaction products. Besides, c-C3H2 carbene have 109 been theoretically calculated to react with a single R-H (R=F, O, N) bond for insertion and also with C=O and C=C bonds for addition.1 Furthermore, reactive functional groups including thiyl group, disulfide linkages are present in amino acids and peptides. These functional groups were reported to be reactive with free radicals like hydroxyl radicals

2 and sulfinyl radicals. It is still unknown if the bi-radical species c-C3H2 could modify these functional groups in their gas-phase ionic forms. In this chapter, the reactivity of c-

C3H2 carbene was studied by the interaction with protonated biomolecules mentioned above for proton-bound reactions as well as other possible gas-phase reactions.

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4.2 Experimental section

Materials

All nucleobases, nucleosides, amino acids, peptides, proteins and lipids were purchased from Sigma Chemical Co. (Sigma-Aldrich, St. Louis, MO). Different concentrations (5-100 μM) of the samples were prepared with methanol/water (9:1, v/v) with 1 % acetic acid added to increase the intensity of protonated species if necessary.

Radical precursors including allylchloride and 1,5-hexadiene were also purchased from

Sigma. Allylchloride was chosen as the major C3H2 carbene precursor unless otherwise specified.

MS System

All conditions were directly adapted from Chapter 3 except for the rf frequency. Two rf frequencies were utilized: the frequency of 950 kHz was employed for m/z 100-500 to observe reactions of amino acids, small peptides, nucleobases and nucleosides, whereas the frequency of 650 kHz allowed high mass range to m/z 1500 to analyze ions of large peptides and proteins. Resonance ejection (q = 0.81) was implemented for MS scan with 110

a dipolar ac signal at 334 and 229 kHz for rf frequency of 950 and 650 kHz, respectively.

4.3 Results and discussion

4.3.1 Nucleobases and nucleosides

The interactions of protonated nucleobases and nucleosides (structures and gas-phase basicities shown in Table 4.1) with c-C3H2 carbene generated by pyrolysis of allylchloride have been investigated to study the carbene modification of DNA/RNA related biomolecules in gas phase.

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Table 4.1 Name, structure, and gas-phase basicity3 (GB) of nucleobases and nucleosides4 Gas- Gas- phase phase Nucleobase Structure Nucleoside Structure basicity basicity (kJ/mol) (kJ/mol)

Adenine 912 Adenosine 957

Guanine 928 Guanosine 961

Cytosine 918 Cytidine 950

Uracil 842 Uridine 917

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Thymine 850 Thymidine 916

For nucleobases of adenine, guanine, and cytosine, their GBs are comparable to that

5 2 of c-C3H2 carbene. Thus, the MS carbene reaction spectra were very similar for these three nucleobases and a significant amount of +38 Da adducts were observed. Taking adenine reaction as an example shown in Figure 4.1a, mass increases from 1 × 38 Da to 3

× 38 Da; which corresponded to mono-, di-, and tri- carbene adducts were observed.

112

Despite adenine having multiple protonation sites,6 the 3 × 38 Da mass increase of m/z

250 implied a primary amine was responsible for protonation in this specific reaction. In the MS3 CID spectrum of Figure 4.1b, a direct loss of 38 Da was obtained when activating the mono-adduct of m/z 174, but the intensity was very low. According to the

7 close GBs of adenine and c-C3H2 carbene, a covalent bond formation should be favored as experimentally demonstrated in Chapter 3. However, the adjacent carbon of the primary amine on adenine is not attached to a hydrogen. The covalent product therefore, would not be able to undergo a loss of 40 Da in the fragmentation pathway as in the cases of amines. The low yield of CID might serve as a piece of indirect evidence of the covalent product formation. As for its corresponding nucleoside adenosine, multiple +38

Da adducts as well as the fragments from the carbene products were obtained in Figure

4.1c. Nucleosides have higher GBs than their corresponding nucleobases.8 Simple complexes should be preferred for adenosine with GB of 957 kJ/mol. In the MS3 CID spectrum of the reaction product at m/z 306, only a small portion of 38 Da loss (m/z 268)

112 appeared as the direct loss of C3H2 in Figure 4.1d, while the principle fragment was the

loss of ribose ring (loss of 132 Da). This result indicated that the hydrogen bond in the proton-bound dimer is stronger than the covalent N-glycosidic bond. The input energy might be able to trigger further rearrangement of the complex to form covalent product.

A very low amount of m/z 136 also appeared and this was probably due to the further activation of m/z 174. Similar reaction phenomena were observed for nucleobases of guanine, cytosine and nucleosides of guanosine, cytidine. All MS2 reaction and MS3 CID spectra are included in Table 4.2.

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Figure 4.1 MS2 carbene reaction spectra of (a) protonated adenine and (c) protonated adenosine; and MS3 CID spectra (b) adenine carbene monoadduct and (d) adenosine carbene monoadduct

9 Uracil and thymine have significantly lower GBs compared to c-C3H2, which makes them harder for the formation of proton-bound dimers. The major product of the protonated molecules for these two species was expected to be a proton-transfer product

+ c-C3H3 , when encountering gas-phase c-C3H2 carbene. Unfortunately, ions of m/z 39 could not be trapped under rf frequency of 950 kHz. Figure 4.2a shows the MS2 reaction spectrum of protonated uracil with c-C3H2 carbene. No adduct was formed with 38 Da

113

mass increase, instead only an intensity decrease of the molecular ions was recorded, probably because of the proton-transfer reaction mentioned above. Although protonated uracil did not form proton-bound dimer with c-C3H2 due to the low GB, its corresponding nucleoside uridine showed such a reaction channel in Figure 4.2b since the ribose ring

10 pushed the electron cloud and thus increased the GB to the same level of c-C3H2. The protonation might locate on the amide amine with two exchangeable hydrogens to induce a di- carbene adduct. The MS3 CID (Figure 4.2c) of the mono-adduct in uridine reaction yielded a similar fragmentation pathway as those of adenosine, guanine, and cytidine.

114

The fact that uracil did not form proton-bound dimer at m/z 151 while uridine-carbene proton-bound dimer yielded m/z 151 during CID, indicates that the formation of proton- bound dimer could be manipulated for low GB compounds with the modification of an easy-to-cleave side chain. For thymine and thymidine, almost identical reactions were recorded in Table 4.2 as uracil and uridine, except that thymidine has a 2’-deoxyribose ring instead of a ribose ring. Thus, the neutral loss observed is 116 Da instead of 132 Da upon activation.

114

Figure 4.2 MS2 carbene reaction spectra of (a) protonated uracil and (b) protonated uridine; and MS3 CID spectrum (c) uridine carbene monoadduct

The reactions between nucleobases, nucleosides and pyrolysis-produced carbene showed that c-C3H2 was able to modify DNA/RNA related gas-phase biomolecular ions.

The reactions of adenine, guanine and cytosine with c-C3H2 tend to form covalent

115 products, yet the products lack dissociation pathway to form fragments during MS3 CID.

Uracil and thymine have GBs that are too low to form proton-bound dimers, however uridine and thymidine are able to form proton-bound dimers and ribose or deoxyribose ring loss upon CID was observed for the product ions. Adenosine, guanosine, and cytidine can form proton-bound dimer with c-C3H2. The proton bound energy is higher than the N-glycosidic bonds and further rearrangements might happen to covert those dimers into covalent products.

Table 4.2 Ion/carbene reactions of nucleobases, nucleosides and c-C3H2 Reactants MS type MS Spectrum

MS2 Reaction

Protonated adenine + c-C3H2

MS3 CID

115

MS2 Reaction

Protonated guanine + c-C3H2

MS3 CID

Protonated cytosine MS2 Reaction + c-C3H2

116

MS3 CID

Protonated uracil + MS2 Reaction c-C3H2

Protonated thymine MS2 Reaction + c-C3H2

MS2 Reaction

Protonated adenosine + c-C3H2

MS3 CID

116

MS2 Reaction

Protonated guanosine + c-C3H2

MS3 CID

Protonated cytidine MS2 Reaction + c-C3H2

117

MS3 CID

MS2 Reaction

Protonated uridine + c-C3H2

MS3 CID

Protonated thymidine MS2 Reaction + c-C3H2

4.3.2 Amino acids, peptides, proteins, and lipids

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4.3.2.1 Protonated amino acids and singly charged peptides

Protonated amino acids containing both amine and carboxylic acid functional groups are expected to react with C3H2 via the proton-bound dimer mechanism proposed in the previous chapter. Compared to the simple amine system studied previously, gas-phase chemistry should be more complicated with these basic building blocks of macro biomolecules in terms of fragmentation pathways during CID.

For basic amino acids of Lys, Arg, and His, their GB values are all higher than c-

2 C3H2, however the MS reaction spectra of those three amino acids all showed abundance

118

38 Da mass increases and new fragmentation pathway in MS3 CID. This was probably due to proton relocation in the basic amino acid ions. The cyclopropenylidene would interact at the proton site which has a similar GB as itself and form a proton-bound dimer.

For instance, the reaction in Figure 2.1a between protonated Lys and c-C3H2 produced mono- and di- adduct of m/z 185 and 223 as well as some other fragment peaks due to the excessive internal energy generated by this reaction. MS3 CID of the reaction product of m/z 185 (Figure 2.1b) yielded all the fragments appeared in the reaction spectrum. A reaction scheme is presented in Figure 2.1c to illustrate a possible fragmentation pathway of the proton-bound dimer. The reaction product of m/z 185 could dissociate into the original reactant protonated Lys of m/z 147 by directly losing c-C3H2 of 38 Da. Thus, protonated Lys further underwent a NH3 loss, generating m/z 130 cyclic ions upon activation, which was a common neutral loss for Lys and other amino acids.11 Another fragmentation pathway was through a covalent product after the carbene reaction. Gas- phase c-C3H2 could be covalently attached to a nitrogen atom and form a new species.

Although the location of the attachment (side chain or N-terminal) is still unknown, the 118

covalent product would undergo a NH3 loss similar to the protonated Lys and yield a cyclic CID product of m/z 168 with an alkyl chain covalently bonded to the amine. This structure could lose 40 Da C3H4 by the attack of the hydrogen on the adjacent carbon as the fragmentation pattern of simple amines in the previous chapter, generating a cyclic iminium of m/z 128. An alternative pathway was for the alkyl chain to grab the hydrogen on the carboxyl acid to further induce a CO2 (44 Da) loss to form a fragment of m/z 124.

Both (1) and (2) pathways were likely to be electrophilic attacks. The loss of 44 Da did not appear in low energy fragmentation pathways12 of amino acids and was highly

119 possible to be related to the carbene chemistry. Indeed, similar loss was also observed in

His/carbene spectrum (Table 1.1).

119

Figure 4.3 (a) MS2 carbene reaction spectrum of protonated Lys, (b) MS3 CID spectrum of Lys/carbene reaction product, and (c) proposed fragmentation pathway of the reaction product.

120

In the case of Arg, CID of protonated Arg (m/z 175) yielded m/z 158 (-H2O), 157 (-

NH3), 140 (-H2O-NH3), 130 (-NH3-CO or -COOH), 116 (-(NH2)2C=NH), and 112 (-H2O-

NH3-CO) (Table 1.1). Therefore, the mono-adduct of m/z 213 in Arg/carbene reaction in

Figure 4.4b yielded: m/z 175 (-C3H2), 169 (-CO2), 152 (-CO2-NH3), 114 (-(NH2)2C=NH -

C3H4), 112 (-C3H2 - H2O-NH3-CO), and 110 (-C3H4 - H2O-NH3-CO) during CID. The fragments of m/z 175 were due to the direct loss of the carbene from the reaction monoadduct; m/z 169 and 152 confirmed the CO2 loss pattern by the carbene similar to

Lys and His; and m/z 114 and 110 were possibly the oxidized products of m/z 116 and

112 (two hydrogen removed) in the Arg CID spectrum respectively. The covalent addition of Arg/carbene reaction might be located at the N-terminal instead of the side guanidine chain according to the CID fragmentation pathways. However, the most favorable protonation site was at the Arg residue in singly charged peptide Met-Arg-Phe-

Ala (MRFA) so that the MS3 CID of the complex of singly charged MRFA showed only dominant fragment at m/z 524 by directly losing C3H2. The alternative N-terminal of Met residue was unlikely to be protonated. 120

Figure 4.4 MS2 carbene reaction spectra of (a) protonated Arg and (c) singly charged MRFA; MS3 CID spectra of (b) Arg/carbene reaction product and (d) MRFA/carbene reaction product.

121

The results of protonated basic amino acids and MRFA interacting with cyclopropenylidene show that although the GBs of the ionic reactants were much higher

3 than C3H2, covalent products could still be formed based on the MS CID fragments, as long as an alternative protonation site existed close enough to the favored charged position in the ionic species which had a suitable GB to form a proton-bound dimer for further rearrangements. Other amino acids were also tested for their modifications with

C3H2 carbene and shown in Table 1.1. A neutral loss of 46 Da (-H2O-CO) was observed

13 for Phe/C3H2 reaction products; a loss of 40 Da (-C3H4) was found for Tyr/C3H2 reaction products; a loss of 17 Da (-NH3) was obtained for Met/C3H2 and Gln/C3H2 reaction products. These results support the formation of a covalent bond.

4.3.2.2 Multiply charged peptides and proteins

Multiply charged peptides could facilitate the C3H2 complex formation through the proton-bound dimer mechanism due to more charges buried inside one ionic species.

Indeed, triply charged Angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu,

121

DRVYIHPFHL) of m/z 433 had up to ten adducts when encountering gas-phase C3H2 by the pyrolysis of allylchloride in Figure 4.5. The number of adducts was related to the number density of C3H2 carbene generated by pyrolysis, which is as a function of the pyrolysis temperature. For 750 degree Celsius, the precursor triply charged ions together with up to four carbene adducts appeared in carbene reaction spectrum (Figure 4.5a), whereas no precursor ions were present anymore for the pyrolysis temperature of 800 and

900 degree Celsius in Figure 4.5b and c. With the higher temperature for pyrolysis, up to ten carbene adducts were observed in Figure 4.5c.

122

Figure 4.5 MS2 reaction spectra of triply charged Angiotensin I with heated allylchoride at (a) 750, (b) 800, and (c) 900 degree Celsius.

Multiply charged protein can also react with the gas-phase carbene species. No mass selection was made to isolate a single ionic population. The purpose of this set of

122 experiment was to evaluate the carbene reactivity towards different charge states. For

Ubiquitin, seven to twelve multiply charged proteins were observed in MS1 full scan in

Figure 4.6a. The protein-carbene complexes corresponding to the eight to eleven charge states were formed in Figure 4.6b. The number of carbene adducts has an increasing trend with the increase of charge states as predicted. The 11+ charge state species yielded approximately 35 carbene adducts based on the difference of m/z values. The lowest addition number observed in this experiment was ca. 23 for the 8+ charge state.

123

Figure 4.6 (a) MS1 spectrum of multiply-charged Ubiquitin, and (b) MS2 spectrum of its reaction with heated allylchoride.

4.3.2.3 Reactive functional groups

Since carbene is a highly reactive species, it would be interesting to investigate if this bi-radical species would interact with relatively reactive functional group such as thiyl group in Cys and disulfide linkages in peptides. Acetyl cysteine with the N-terminal blocked was selected to test the reactivity of thiyl group and c-C3H2 carbene, which was

123 designed to minimize the reaction of the protonated amine site. The very low yield of 38

Da mass increase from the protonated species in Figure 4.7a, proved that thiyl group had limited reactivity when encountering gas-phase c-C3H2 carbene. The mass increase was most likely due to the complex formation with the protonated amide, similar to the cases in thymine and uracil.

Disulfide linkage is an important posttranslational modification to construct three dimensional structure in proteins. A simple disulfide linked di- amino acid cystine was used in its protonated form to interact with c-C3H2 carbene. Despite the abundant 38 Da

124 mass increases in the reaction spectrum (Figure 4.7b), there was no ion observed from m/z 80-160 (cysteine thiyl radical ion: m/z 121, perthiyl radical ion: m/z 153), which indicated no disulfide cleavage occurred during the gas-phase carbene reaction. Moreover, using singly charged oxidized glutathione (single chain sequence: γ-Glu-Cys-Gly, γECG, disulfide linked), the m/z shift still appeared to be 38 Da, however, no sign of disulfide cleavage was observed in Figure 4.7c.

124

Figure 4.7 MS2 carbene reaction spectra of (a) protonated acetyl cysteine, (b) protonated cystine, and (c) singly charged oxidized glutathione.

Unsaturated phospholipids play an important role in biological functions.14 It was theoretically calculated and reported that c-C3H2 can react with hydroxyl group for single bond insertion,1a and neutral unsaturated compounds for double bond addition.1c, 1d

Nevertheless, there has yet to be any experimental evidence for the mentioned

125 neutral/carbene or ion/carbene reactions. Choline was then used to test the reactivity of c-

C3H2 with hydroxyl group. The advantage of gas-phase choline ion is that it has a fixed charge to eliminate the proton-bound dimer reaction. In the reaction spectrum of Figure

4.8a, no mass shift was observed for choline ions, inferring insertion reactions did not occur under the current experimental conditions between choline and c-C3H2.

Furthermore, the sodiated lysophosphatidylcholine (LPC) with an eighteen-carbon acetyl chain and two double bonds in the 9th and 12th positions (18:2∆9, 12) was generated and isolated to interact c-C3H2 carbene in Figure 4.8b. The lack of ions at m/z 590 indicated the gas-phase cyclopropenylidene did not undergo double bond insertion in the LPC studied.

125

Figure 4.8 MS2 reaction spectra of (a) hydroxyl group containing choline and (b) double bond containing LPC (18:2) sodium adduct. No reaction product was observed.

The absence of reactivity of gas-phase c-C3H2 towards neutral thiyl group, disulfide linkages, hydroxyl group, and double bonds based on the results of Figure 4.7 and Figure

4.8, suggested that the gas-phase chemistry of ion/c-C3H2 was mainly driven by the

126 proton and the mechanism proposed in the previous chapter. The theoretically calculated reaction pathway might not occur or be totally shadowed because of the high GB of the cyclic carbene. All MS2 reaction and MS3 CID spectra are attached in Table 1.1 for all the biomolecular ions studied in this section.

Table 4.3 Ion/carbene reactions of amino acids, peptides, protein and c-C3H2 Reactant and GB MS type MS Spectrum

MS2 Reaction Protonated Lysine

951 kJ/mol MS3 CID

Protonated Histidine

MS2 Reaction

950 kJ/mol 126

MS2 Reaction

Protonated Arginine

MS3 CID

1007 kJ/mol

MS2 CID

127

Protonated Leucine

MS2 Reaction

881 kJ/mol Protonated Methionine

MS2 Reaction

902 kJ/mol

Protonated MS2 Reaction Phenylalanine

3 889 kJ/mol MS CID

MS2 Reaction Protonated Tyrosine

127

892 kJ/mol MS3 CID

MS2 Reaction Protonated Glutamine

900 kJ/mol MS3 CID

128

MS2 Reaction Protonated Proline methyl ester

MS3 CID

Proline methyl ester MS2 Reaction sodium adduct

MS3 CID

Protonated Cysteine methyl ester MS2 Reaction

Protonated Acetyl

128 Cysteine

MS2 Reaction

Protonated Acetyl Cysteine methyl ester

MS2 Reaction

Protonated Cystine MS2 Reaction with heated allylchloride

129

MS3 CID

MS2 Reaction with heated propargylbromide

MS2 Reaction with heated pentane

MS2 Reaction with heated d12- pentane

MS2 Reaction with heated dichloromethane

129

Singly charged MS2 Reaction Oxidized glutathione γ-ECG | γ-ECG Doubly charged MS2 Reaction

Singly charged Met-Arg-Phe-Ala MS2 Reaction MRFA

130

MS3 CID

MS2 Reaction Low amount of C3H2

Triply charged Angiotensin I MS2 Reaction Asp-Arg-Val-Tyr-Ile- Medium amount His-Pro-Phe-His-Leu of C3H2 DRVYIHPFHL

MS2 Reaction High amount of C3H2

MS1

Ubiquitin

130

MS2 Reaction

Choline MS2 Reaction

Lysophosphatidylcholine

LPC(18:2) sodium 2 adduct MS Reaction

131

4.4 Conclusion

The interaction between biomolecular ions and c-C3H2 have been investigated.

Proton-bound dimers were formed for protonated nucleobases and nucleosides including adenine, guanine, cytosine, adenosine, guanosine, cytidine, thymidine, and uridine. The hydrogen bond energy in the proton-bound dimer was determined to be higher than the

N-glycosidic bonds in all nucleosides. The GB values for uracil and thymine are too low for the complex to form. Protonated basic amino acids were shown to generate covalent reaction product with c-C3H2 carbene by relocating the proton to a charged site of a similar GB. Multiply charged peptides and proteins have a wide range of adduct distribution with different amounts of c-C3H2 generated by various pyrolysis temperatures. However, the gas-phase reactivity of c-C3H2 carbene was mainly driven by the charge and high GB of this species. The bi-radical carbene species had limited or no modification for free thiyl group and disulfide linkages. Furthermore, the calculated reactivity of double bond addition and single bond insertion did not occur with biomolecular ions such as choline and LPC. The proton-bound dimer reaction remained 131

the dominant channel for all the biomolecular ion/c-C3H2 reactions.

132

4.5 References

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6. de Meijere, A.; Kozhushkov, S. I., An evolving multifunctional molecular building block: Bicyclopropylidene. Eur. J. Org. Chem. 2000, (23), 3809-3822. 7. Marek, I.; Simaan, S.; Masarwa, A., Enantiomerically enriched cyclopropene derivatives: Versatile building blocks in asymmetric synthesis. Angew. Chem., Int. Ed. 2007, 46 (39), 7364-7376. 8. Dookeran, N. N.; Yalcin, T.; Harrison, A. G., Fragmentation reactions of protonated alpha-amino acids. J. Mass Spectrom. 1996, 31 (5), 500-508. 9. Yao, C.; Syrstad, E. A.; Tureček, F., Electron transfer to protonated beta-alanine N-methylamide in the gas phase: An experimental and computational study of dissociation energetics and mechanisms. J. Phys. Chem. A 2007, 111 (20), 4167-4180.

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10. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; ; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J., Gaussian 09, Revision A. 02; Gaussian, Inc., Wallingford, CT 2009. 11. Marion, N.; Nolan, S. P., Well-Defined N-Heterocyclic Carbenes-Palladium(II) Precatalysts for Cross-Coupling Reactions. Acc. Chem. Res. 2008, 41 (11), 1440-1449. 12. Mebel, A. M.; Jackson, W. M.; Chang, A. H. H.; Lin, S. H., Photodissociation dynamics of propyne and allene: A view from ab initio calculations of the C3Hn (n=1-4) species and the isomerization mechanism for C3H2. J. Am. Chem. Soc. 1998, 120 (23), 5751-5763. 13. Vazquez, J.; Harding, M. E.; Gauss, J.; Stanton, J. F., High-Accuracy Extrapolated ab Initio Thermochemistry of the Propargyl Radical and the Singlet C3H2 Carbenes. J. Phys. Chem. A 2009, 113 (45), 12447-12453. 14. Wass, D. F.; Haddow, M. F.; Hey, T. W.; Orpen, A. G.; Russell, C. A.; Wingad, R. L.; Green, M., Cyclopropenylidene carbene ligands in palladium C-C coupling catalysis. Chem. Commun. 2007, (26), 2704-2706.

133

VITA

134

VITA

Ziqing Lin was born in 1984 in Shanghai, China. In the summer of 2003, he was admitted to Tsinghua University in Beijing, China, and majored in Chemistry. There, he obtained a solid knowledge and training in analytical, inorganic, organic, and physical chemistry. As an undergraduate junior, Ziqing had an undergraduate research program with Prof. Xinrong Zhang in Key Laboratory for Atomic and Molecular Nanosciences of the Education Ministry, Department of Chemistry, Tsinghua University. In 2006, he joined Prof. Zhang’s lab for his senior design and encountered mass spectrometry, where he found the field fascinating, that analytical chemistry especially mass spectrometry could be an essential key to monitor and solve biomedical problems. In 2007, Ziqing was admitted to Department of Chemistry, Tsinghua University for a 3-year Master’s program

134 after his bachelor degree. He continued to work on applications of ambient mass

spectrometry under the supervision of Prof. Zhang and developed a rapid screening methodology for clenbuterol, an abused drug and speciation for 8 organic and inorganic arsenic species. Besides his research as an analyst, Ziqing learned mechanical drawing, and made compartments to modify the atmospheric pressure interface for mass spectrometers as well as helping his colleagues design instrumentational devices. He also drew an amount of cover pictures for scientific journals. Therefore, Ziqing gradually grew into a combination of scientist, engineer, and artist. In 2010 after obtaining his

Master’s degree in analytical chemistry, Ziqing decided to come to the United States and

135 pursue his PhD degree in Weldon School of Biomedical Engineering at Purdue

University. He joined Prof. Zheng Ouyang’s group and further worked on instrumentation and application of mass spectrometry for gas-phase biomolecular ion reactions, which was a joint program of both science and engineering. After building his own mass spectrometry system, he discovered for the first time a cyclopropenylidene reaction with protonated alkyl amines, amino acids and peptides. Ziqing also collaborated on the development of long-distance ion-transferring tube and other ambient mass spectrometry applications.

135

PUBLICATIONS

136

PUBLICATIONS

Journals

1. Lin, Z.; Tan, L.; Yang, Y.; Dai, M.; Tureček, F.; Ouyang, Z.; Xia, Y. Gas-phase

reactions of C3H2 with protonated alkyl amines, J. Am. Chem. Soc. In preparation.

2. Lin, Z.; Tan, L.; Garimella, S.; Li, L.; Chen, T.; Xu, W.; Xia, Y.; Ouyang, Z.

Characterization of a DAPI-RIT-DAPI system for gas-phase ion/molecule and ion/ion

reactions, J. Am. Soc. Mass Spectrom. 2014, 25, 48-56.

3. Chen, C.; Lin, Z.; Garimella, S.; Zheng, L.; Shi, R.; Cooks, R. G.; Ouyang, Z.

Development of a mass spectrometry sampling probe for endoscopic analysis, Anal.

Chem. 2014, 85, 11843-11850.

4. Lin, Z.; Zhao, M.; Zhang, S.; Yang, C.; Zhang, X. In situ arsenic speciation on solid

136 surfaces by desorption electrospray ionization tandem mass spectrometry, Analyst

2010, 135, 1268-1275.

5. Liu, Y. ; Ma, X.; Lin, Z.; He, M.; Han, G.; Yang, C.; Xing, Z.; Zhang, S.; Zhang, X.

Imaging mass spectrometry with a low-temperature plasma probe for the analysis of

works of art, Angew. Chem.-Int. Edit. 2010, 49, 4435-4437

6. Liu, Y.; Lin, Z.; Zhang, S.; Yang, C.; Zhang, X. Rapid screening of active ingredients

in drugs by mass spectrometry with low-temperature plasma probe, Anal. Bioanal.

Chem. 2009, 395, 591-599.

137

7. Ma, X.; Zhang, S.; Lin, Z.; Liu, Y.; Xing, Z.; Yang, C.; Zhang, X. Real-time

monitoring of chemical reactions by mass spectrometry utilizing a low-temperature

plasma probe, Analyst 2009, 134, 1863-1867.

8. Lin, Z.; Zhang, S.; Zhao, M.; Yang, C.; Chen, D.; Zhang, X. Rapid screening of

clenbuterol in urine samples by desorption electrospray ionization tandem mass

spectrometry, Rapid Commun. Mass Spectrom. 2008, 22, 1882-1888.

9. Ma, X.; Zhao, M.; Lin, Z.; Zhang, S.; Yang, C.; Zhang, X. Versatile platform

employing desorption electrospray ionization mass spectrometry for high-throughput

analysis, Anal. Chem. 2008, 80, 6131-6136.

Conference Oral and Poster Presentations

1. Lin, Z.; Tan, L.; Yang, Y.; Dai, M.; Tureček, F.; Ouyang, Z.; Xia, Y. Reactions of

Biomolecule Ions with Pyrolysis-formed Carbene, Poster TP353, 62nd ASMS Annual

Conference on Mass Spectrometry and Allied Topics, Baltimore, MD, Jun 17th, 2014.

137 2. Lin, Z.; Tan, L.; Ouyang, Z.; Xia, Y. Instrumentation and studies for gas-phase

ion/radical reactions, Poster, 26th ASMS Sanibel Conference on Mass Spectrometry,

Clearwater Beach, Fl, US, Jan 30th-Feb 2nd, 2014.

3. Lin, Z.; Tan, L.; Chen, T.; Li, L.; Xia, Y.; Ouyang, Z. Development of a mass

spectrometer for gas phase ion/radical reactions, Oral TOA1450, 61st ASMS Annual

Conference on Mass Spectrometry and Allied Topics, Minneapolis, MN, US, Jun 11th,

2013.

4. Lin, Z.; Garimella, S.; Li, L; Chen, T.; Xu, W.; Xia, Y.; Ouyang, Z. A DAPI-RIT-

DAPI system for in-trap gas phase reactions, Oral TOC850, 60th ASMS Annual

138

Conference on Mass Spectrometry and Allied Topics, Vancouver, BC, Canada, May

22nd, 2012.

5. Chen, C.; Lin, Z.; Garimella, S.; Cooks, R. G.; Ouyang, Z. Development of an

endoscopic DESI sampling probe, Poster TP034, 59th ASMS Annual Conference on

Mass Spectrometry and Allied Topics, Denver, CO, Jun 7th, 2011.

138