principles of mass spectrometry applied to … fileusing anion photoelectron spectroscopy in the gas...

PRINCIPLES OF MASSSPECTROMETRYAPPLIED TOBIOMOLECULES

Edited by

JULIA LASKIN, PhD

Pacific Northwest National Laboratory

Richland, Washington

CHAVA LIFSHITZ, PhD

The Hebrew University

Jerusalem, Israel

A JOHN WILEY & SONS, INC., PUBLICATION

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WILEY-INTERSCIENCE SERIES IN MASS SPECTROMETRY

Series Editors:

Dominic M. Desiderio

Departments of Neurology and Biochemistry

University of Tennessee Health Science Center

Nico M. M. Nibbering

Vrije Universiteit Amsterdam, The Netherlands

John R. de Laeter � Applications of Inorganic Mass SpectrometryMichael Kinter and Nicholas E. Sherman � Protein Sequencing and Identification

Using Tandem Mass Spectrometry

Chhabil Dass, Principles and Practice of Biological Mass Spectrometry

Mike S. Lee � LC/MS Applications in Drug DevelopmentJerzy Silberring and Rolf Eckman � Mass Spectrometry and Hyphenated Techni-

ques in Neuropeptide Research

J. Wayne Rabalais � Principles and Applications of Ion Scattering Spectrometry:Surface Chemical and Structural Analysis

Mahmoud Hamdan and Pier Giorgio Righetti � Proteomics Today: Protein Assess-ment and Biomarkers Using Mass Spectrometry, 2D Electrophoresis, and

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Igor A. Kaltashov and Stephen J. Eyles � Mass Spectrometry in Biophysics:Conformation and Dynamics of Biomolecules

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Julia Laskin and Chava Lifshitz � Principles of Mass Spectrometry Applied toBiomolecules


Edited by

JULIA LASKIN, PhD

Pacific Northwest National Laboratory

Richland, Washington

CHAVA LIFSHITZ, PhD

The Hebrew University

Jerusalem, Israel

A JOHN WILEY & SONS, INC., PUBLICATION

Copyright # 2006 by John Wiley & Sons, Inc. All rights reserved

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Library of Congress Cataloging-in-Publication Data:

Principles of mass spectrometry applied to biomolecules/edited by Julia Laskin, Chava Lifshitz.

p. cm.

Includes bibliographical references and index.

ISBN-13 978-0-471-72184-0 (cloth)

ISBN-10 0-471-72184-0 (cloth)

1. Mass spectrometry. 2. Biomolecules–Analysis. I. Laskin, Julia, 1967-II. Lifshitz, Chava.

QP519.9.M3P77 2006

5430.65–dc22 2006043900

Printed in the United States of America

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This book is dedicated to the memory of Chava Lifshitz—one of the pioneers of the

field of gas-phase ion chemistry and fundamental mass spectrometry—a great

scientist, an excellent mentor, and a good friend.

—JULIA LASKIN

CONTENTS

CONTRIBUTORS xi

PREFACE xv

PART I STRUCTURES AND DYNAMICS OF GAS-PHASEBIOMOLECULES 1

1 Spectroscopy of Neutral Peptides in the Gas Phase:Structure, Reactivity, Microsolvation,Molecular Recognition 3

Markus Gerhards

2 Probing the Electronic Structure of Fe–S Clusters:Ubiquitous Electron Transfer Centers in MetalloproteinsUsing Anion Photoelectron Spectroscopy in the Gas Phase 63

Xin Yang, Xue-Bin Wang, You-Jun Fu, and Lai-Sheng Wang

3 Ion–Molecule Reactions and H/D Exchange for StructuralCharacterization of Biomolecules 119

M. Kirk Green and Carlito B. Lebrilla

4 Understanding Protein Interactions and TheirRepresentation in the Gas Phase of the Mass Spectrometer 147

Frank Sobott and Carol V. Robinson

vii

5 Protein Structure and Folding in the Gas Phase:Ubiquitin and Cytochrome c 177

Kathrin Breuker

6 Dynamical Simulations of Photoionization of SmallBiological Molecules 213

Dorit Shemesh and R. Benny Gerber

7 Intramolecular Vibrational Energy Redistributionand Ergodicity of Biomolecular Dissociation 239

Chava Lifshitz

PART II ACTIVATION, DISSOCIATION, AND REACTIVITY 277

8 Peptide Fragmentation Overview 279

Vicki H. Wysocki, Guilong Cheng, Qingfen Zhang,

Kristin A. Herrmann, Richard L. Beardsley,

and Amy E. Hilderbrand

9 Peptide Radical Cations 301

Alan C. Hopkinson and K. W. Michael Siu

10 Photodissociation of Biomolecule Ions: Progress, Possibilities,and Perspectives Coming from Small-Ion Models 337

Robert C. Dunbar

11 Chemical Dynamics Simulations of Energy Transfer andUnimolecular Decomposition in Collision-Induced Dissociation(CID) and Surface-Induced Dissociation (SID) 379

Asif Rahaman, Kihyung Song, Jiangping Wang,

Samy O. Meroueh, and William L. Hase

12 Ion Soft Landing: Instrumentation, Phenomena,and Applications 443

Bogdan Gologan, Justin M. Wiseman,

and R. Graham Cooks

13 Electron Capture Dissociation and OtherIon–Electron Fragmentation Reactions 475

Roman Zubarev

14 Biomolecule Ion–Ion Reactions 519

Scott A. McLuckey

viii CONTENTS

PART III THERMOCHEMISTRY AND ENERGETICS 565

15 Thermochemistry Studies of Biomolecules 567

Chrys Wesdemiotis and Ping Wang

16 Energy and Entropy Effects in Gas-PhaseDissociation of Peptides and Proteins 619

Julia Laskin

INDEX 667

CONTENTS ix

CONTRIBUTORS

Richard L. Beardsley, Department of Chemistry, Box 210041, University of

Arizona, 1306 East University Avenue, Tucson, AZ 85721-0041

Kathrin Breuker, Institute of Organic Chemistry and Center for Molecular

Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 52a, A-6020

Innsbruck, Austria

Guilong Cheng, Department of Chemistry, Box 210041, University of Arizona,

1306 East University Avenue, Tucson, AZ 85721-0041

R. Graham Cooks, Department of Chemistry, Purdue University, 560 Oval Drive,

West Lafayette, IN 47907-2038

Robert C. Dunbar, Chemistry Department, Case Western Reserve University,

Cleveland, OH 44106

You-Jun-Fu, Department of Physics, Washington State University, 2710 University

Drive, Richland, WA 99352; W. R. Wiley Environmental Molecular Sciences

Laboratory and Chemical Sciences Division, Pacific Northwest National

Laboratory, MS K8-88, P.O. Box 999, Richland, WA 99352

R. Benny Gerber, Department of Chemistry, University of California, Irvine, CA

92697; Department of Physical Chemistry and the Fritz Haber Research Center,

The Hebrew University, Jerusalem 91904, Israel

Markus Gerhards, Heinrich-Heine Universität Düsseldorf, Institut für Physika-

lische Chemie I, Universitätstrasse 26.33.O2, 40225 Düsseldorf, Germany

xi

Bogdan Gologan, Department of Chemistry, Purdue University, 560 Oval Drive,

West Lafayette, IN 47907-2038

M. Kirk Green, McMaster Regional Centre for Mass Spectrometry, Department of

Chemistry, McMaster University, Hamilton, Canada

William L. Hase, Department of Chemistry and Biochemistry, Texas Tech

University, Lubbock, TX 79409-1061

Kristin A. Herrmann, Department of Chemistry, Box 210041, University ofArizona, 1306 East University Avenue, Tucson, AZ 85721-0041

Amy E. Hilderbrand, Department of Chemistry, Box 210041, University of

Arizona, 1306 East University Avenue, Tucson, AZ 85721-0041

Alan C. Hopkinson, Centre for Research in Mass Spectrometry and the Department

of Chemistry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J

1P3

Julia Laskin, Fundamental Sciences Division, Pacific Northwest National

Laboratory, P.O. Box 999 K8-88, Richland, WA 99352

Carlito B. Lebrilla, Department of Chemistry, University of California, Davis, CA

95616

Chava Lifshitz, Department of Physical Chemistry and The Farkas Center for Light

Induced Processes, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

Scott A. McLuckey, Department of Chemistry, Purdue University, 560 Oval Drive,West Lafayette, IN 47907-2084

Samy O. Meroueh, Department of Chemistry and Biochemistry, University of

Notre Dame, Notre Dame, IN 46556-5670

Asif Rahaman, Department of Chemistry and Biochemistry, Texas Tech University,

Lubbock, TX 79409-1061

Carol V. Robinson, The University Chemical Laboratory, University of Cambridge,

Lensfield Road, Cambridge CB2 1EW, United Kingdom

Dorit Shemesh, Department of Physical Chemistry and The Fritz Haber Research

Center, The Hebrew University, Jerusalem 91904, Israel

K. W. Michael Siu, Centre for Research in Mass Spectrometry and the Departmentof Chemistry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J

1P3

Frank Sobott, Structural Genomics Consortium, University of Oxford, BotnarResearch Centre, Oxford OX3 7LD, United Kingdom

Kihyung Song, Department of Chemistry, Korea National University of Education,

Chongwon, Chungbuk 363-791, Korea

xii CONTRIBUTORS

Jiangping Wang, Department of Chemistry, Wayne State University, Detroit, MI

48202

Lai-Sheng Wang, Department of Physics, Washington State University, 2710

University Drive, Richland, WA 99352; W. R. Wiley Environmental Molecular

Sciences Laboratory and Chemical Sciences Division, Pacific Northwest

National Laboratory, MS K8-88, P.O. Box 999, Richland, WA 99352

Ping Wang, Department of Chemistry, The University of Akron, Akron, OH 44325

Xue-Bin Wang, Department of Physics, Washington State University, 2710

University Drive, Richland, WA 99352; W. R. Wiley Environmental Molecular

Sciences Laboratory and Chemical Sciences Division, Pacific Northwest

National Laboratory, MS K8-88, P.O. Box 999, Richland, WA 99352

Chrys Wesdemiotis, Department of Chemistry, The University of Akron, Akron,

OH 44325

Justin M. Wiseman, Department of Chemistry, Purdue University, 560 Oval Drive,West Lafayette, IN 47907-2038

Vicki H. Wysocki, Department of Chemistry, Box 210041, University of Arizona,


Xin Yang, Department of Physics, Washington State University, 2710 University

Drive, Richland, WA 99352; W. R. Wiley Environmental Molecular Sciences

Laboratory and Chemical Sciences Division, Pacific Northwest National

Laboratory, MS K8-88, P.O. Box 999, Richland, WA 99352

Qingfen Zhang, Department of Chemistry, Box 210041, University of Arizona,


Roman Zubarev, Laboratory for Biological and Medical Mass Spectrometry

Uppsala University, Box 583, Uppsala S-751 23, Sweden

CONTRIBUTORS xiii

PREFACE

The introduction of biological molecules into the gas phase by matrix-assisted laser

desorption/ionization (MALDI) and electrospray ionization (ESI) has led to a

revolution in biological mass spectrometry. The analytical aspects are a success

story. Molecular weights can be determined with a high precision, peptide

sequencing is now done with great success, and even higher-order structures of

peptides and proteins can be accessed using mass spectrometry. Exceptionally high

sensitivity, high mass resolution, and inherent speed are the key factors that

positioned mass spectrometry at the forefront of analytical techniques for

identification and characterization of biomolecules.

This success is based largely on the principles of mass spectrometry that have

been developed since the mid-1970s for small organic molecules. However, studies

of biomolecules in the gas phase have also revealed a number of challenges

associated with the flexibility and the size of these species. For example, it

was difficult to achieve efficient fragmentation of large molecules using traditional

mass spectrometric approaches. Understanding of fundamental limitations of the

existing ion activation techniques resulted in development of novel analytical

approaches for studying fragmentation of large molecules in the gas phase.

Improved identification of biomolecules in real-world applications is facilitated by

understanding of their fragmentation mechanisms and the effect of the primary and

the secondary structure on the observed fragmentation patterns.

Because of the large size, conformational flexibility, and the ability of

biomolecules to hold multiple charges, studies of biomolecular gas-phase ion

chemistry have opened a number of new and exciting areas of research. Multiply

charged biomolecules are excellent targets for studying ion–ion chemistry and

processes following capture of low-energy electrons. Various approaches are being

xv

developed to gain phenomenological understanding of the formation and

fragmentation of hydrogen-rich radical cations, molecular radical cations, and

radical anions of peptides and proteins. Development of new approaches for

studying thermochemistry of gas-phase biomolecules and their dissociation

energetics is at the forefront of the field. Vibrational spectroscopy of biomolecular

ions is another area of research that is currently undergoing an explosive growth. In

parallel, new high-resolution spectroscopic techniques have been successfully

applied to larger systems, providing feedback to mass spectrometric studies.

Reactivity of mass-selected biomolecules with solid targets has a potential for

preparation of novel surfaces relevant for a variety of applications in biology and

biotechnology.

In addition, there are several basic aspects related to the physics of the various

problems that have remained unanswered. For example, the question of ergodicity

and/or statistical versus nonstatistical behavior in the breakup of biomolecules has

been raised in connection with several methods, including electron capture

dissociation (ECD) or photodissociation. The old questions that were raised many

years ago concerning organic molecules are again at the forefront—do gas phase

biomolecules undergo intramolecular vibrational redistribution (IVR) prior to

dissociation? Are all vibrational modes involved in IVR? Is there site selectivity and

charge-directed reactivity? The mere fact that a large protein fragments on the short

timescale of mass spectrometry, which is an absolute necessity in terms of analysis

and sequencing, is somewhat surprising in view of our previous knowledge of

dissociation of relatively small organic molecules in the gas phase and its description

using statistical theories [Rice–Ramsperger–Kassel–Marcus/quasiequilibrium

theory (RRKM/QET) and the like].

This book is a collection of reviews on fundamental aspects underlying mass

spectrometry of biomolecules. The various selected topics have been arranged in

three parts: (1) structures and dynamics of gas-phase biomolecules; (2) activation,

dissociation, and reactivity; and (3) thermochemistry and energetics.

Fundamental mass spectrometry has always been strongly linked to a variety of

gas-phase spectroscopic techniques, which provide unique insights on the structure

and dynamics of ions and molecules in the gas phase. High-resolution UV and IR

spectroscopy discussed in Chapter 1 allows study of the structure and dynamics of

individual conformers of neutral biomolecules, exploring the effect of the solvent on

the intrinsic properties of these molecules, and molecular recognition by examining

the behavior of gas-phase clusters of biomolecules. Chapter 2 gives an example of

high-resolution photodetachment phoelectron spectroscopy studies of electron

transfer in iron–sulfur (Fe–S) clusters. In particular, this technique is used to explore

the effect of solvents and protein environment on the electronic properties of the

cubane-type [4Fe–4S] cluster—the most common agent for electron transfer and

storage in metalloproteins.

Ion–molecule reactions and H/D (hydrogen/deuterium) exchange studies have

traditionally been used in mass spectrometry for structure determinations. Chapter 3

gives an overview of the application of these techniques to studies of structures and

conformations of gas-phase biomolecules. While spectroscopic techniques are

xvi PREFACE

currently limited to relatively small systems, mass spectrometry has been used to

investigate quaternary structures of large protein complexes. Experimental

approaches utilized in such studies are summarized in Chapter 4. Protein structures

and folding in the gas phase is discussed in Chapter 5. Understanding protein

dynamics in the absence of solvent—the driving force and the timescale of protein

folding in the gas phase—is important for separating the effect of solvent from the

effect of the intrinsic properties of proteins on their dynamics in solution.

The dynamics of the intramolecular vibrational energy redistribution (IVR) in

gas-phase biomolecules is discussed in Chapters 6 and 7. Classical trajectory

simulations using semiempirical PM3 potential energy surfaces described in

Chapter 6 are instrumental for understanding ultra fast dynamics following

photoionization of biomolecules and the validity of statistical theories of

dissociation of these large floppy molecules. Studies of gas-phase ion chemistry

of peptides and proteins revealed a variety of very interesting phenomena, some of

which (e.g., electron capture dissociation and photodissociation) were described as

nonergodic processes that circumvent IVR. The pros and cons of IVR and ergodic

behavior in biomolecules based on the available experimental findings are discussed

in Chapter 7.

Gas-phase fragmentation of protonated peptides is an important prerequisite for

peptide and protein identification using tandem mass spectrometry (MS/MS).

Understanding mechanistic aspects of peptide fragmentation as a function of peptide

sequence and conformation summarized in Chapter 8 plays a central role in the

interpretation of MS/MS spectra and refining strategies for database searching. Most

mass spectrometric studies utilize closed-shell biomolecules (protonated or

cationized on metals) generated using soft ionization techniques. Formation and

dissociation of peptide radical cations described in Chapter 9 is a new rapidly

growing field in gas-phase ion chemistry of biomolecules. These ions are formed by

gas-phase fragmentation of complexes of the corresponding neutral peptide with

transition metals and various organic ligands.

Collisional activation and multiphoton excitation are conventionally used for

identification of biomolecules in a variety of mass spectrometric applications.

Current status of multiphoton excitation, spectroscopy, and photodissociation of gas-

phase biomolecules is summarized in Chapter 10. Chapter 11 presents classical

trajectory simulations of the energy transfer in collisions of ions with atomic neutrals

and surfaces. The phenomena observed following ion–surface collisions and the

instrumentation involved in such studies are presented in Chapter 12 with particular

emphasis on soft landing of biological molecules on a variety of surfaces. Soft

landing can be utilized for a very specific modification of surfaces using a beam of

mass-selected ions of any size and composition or for separating and preparing

biomolecules on substrates in pure form for subsequent analysis.

Another method of ion activation in biological mass spectrometry relies on

capture of low-energy electrons by multiply charged ions. Electron capture

dissociation (ECD), discussed in Chapter 13, opens up a variety of unique

dissociation pathways and provides information on the structure of the ion that is

complementary to collisional or multiphoton excitation. Chapter 14 presents the

PREFACE xvii

fundamental principles of ion–ion chemistry of biomolecules. Ion–ion reactions

provide a means of manipulating charge states of multiply charged peptides and

proteins. Charge reduction by reactions of multiply charged biomolecules with

singly charged ions of opposite polarity has developed as a powerful tool for

structural elucidation of peptides and proteins.

Mass spectrometry has been widely utilized for thermochemical determinations.

However, studying thermochemistry and dissociation energetics of peptides and

proteins is challenging because most of the well-developed experimental approaches

that have been successfully employed in the studies of small and medium-size ions

are simply not applicable to the fragmentation of large molecules. Chapter 15

presents an overview of mass spectrometric approaches that have been utilized for

thermochemical determinations of biomolecules and discusses the current status and

limitations of these techniques, focusing on determination of proton affinities and

alkali metal affinities of biomolecules. Chapter 16 describes the experimental

approaches developed for studying the energetics and entropy effects in peptide and

protein dissociation reactions.

Finally, we would like to acknowledge the authors of the chapters, who have

invested a considerable amount of time and effort and prepared high-quality reviews

for this book. Special thanks go to Jean Futrell for his generous help on various

stages of this project and insightful feedback on the contents of several chapters. We

are also thankful to many other colleagues who provided their comments and

suggestions on the contents of this book.

JULIA LASKIN AND CHAVA LIFSHITZ

xviii PREFACE

PART I

STRUCTURES AND DYNAMICS OFGAS-PHASE BIOMOLECULES

1SPECTROSCOPY OF NEUTRALPEPTIDES IN THE GAS PHASE:STRUCTURE, REACTIVITY,MICROSOLVATION, MOLECULARRECOGNITION

MARKUS GERHARDSHeinrich-Heine Universität Düsseldorf

Institut für Physikalische Chemie I

Düsseldorf, Germany

1.1. Introduction and Historical Background

1.2. Experimental Setups and Methods

1.2.1. Laser Spectroscopic Methods and Microwave Spectroscopy

1.2.2. Some Experimental Setups: Mass Spectrometry, Double-Resonance

Spectroscopy, and Sources

1.3. Spectroscopy on Selected Amino Acid Model Systems

1.4. Double-Resonance and Microwave Spectroscopy on Amino Acids

1.4.1. Phenylalanine

1.4.2. Tryptophan

1.4.3. Applications of Microwave Spectroscopy

1.5. Spectroscopic Analysis of Peptide Structures

1.6. Molecular Recognition

1.7. Calculations and Assignment of Vibrational Frequencies

1.8. Summary and Outlook

Principles of Mass Spectrometry Applied to Biomolecules, edited by Julia Laskin and Chava LifshitzCopyright # 2006 John Wiley & Sons, Inc.

3

1.1. INTRODUCTION AND HISTORICAL BACKGROUND

As reported in previous chapters of the book, it has been a great challenge to transfer

large molecules in the gas phasewithout dissociation. The investigations focus on a pure

mass spectrometric analysis, but no spectroscopic information on the analyzed species is

available. To obtain more information on the energy of different electronic states as well

as the structure and dynamical changes of the investigated isolated species, the pure

mass spectrometry has to be combined with different spectroscopic techniques. The

motivation is strongly triggered by the following questions:

(1) What are the driving forces for protein folding or aggregation of peptides?

(2) How does solvation change the secondary structure of peptides, and how can

this process be influenced, i.e. in our investigations can we perform experiments

on mass-selected peptides and can we add, for instance, one water molecule

after the other in order to determine how the structures will change?

By answering these questions on a molecular level, we may contribute to

explanations of how structures and dynamics of peptides can be understood or

predicted. The main focus of this chapter is a review on the most important

combined spectroscopic and mass spectrometric analyses. This chapter focuses only

on neutral amino acids and peptides; the spectroscopic investigation of ionic species

is another rapidly growing field and will not be discussed here.

As mentioned in other chapters, large charged molecules can be transferred into

the gas phase by applying MALDI (Karas and Hillenkamp 1988), ESI (Fenn et al.

1989), or LILBID (laser-induced liquid beam ion description) (Kleinekofort et al.

1996) and other sources. Neutral molecules can be transferred by heating sources,

but in the case of pure amino acids or peptides, the molecules can easily fragment by

elimination of CO2. Different sources for transferring neutral species into the gas

phase are discussed in this chapter. A major breakthrough was the introduction of laser

desorption sources (see Section 1.2) in combination with supersonic cooling and laser

ionization (of the neutral desorbed species). The combination of this pure mass

spectrometry on selected neutral species (which are ionized for detection as cations)

with spectroscopic techniques was triggered by the pioneering work of Levy and

coworkers (Cable et al. 1987, 1988a,b; Rizzo et al. 1985, 1986b). Starting from the

analysis of amino acids by a combination of laser desorption and fluorescence

spectroscopy or resonant multiphoton ionization, the Levy group increases the size of

the investigated species up to tripeptides (Cable et al. 1987, 1988a,b). The

spectroscopic results yield information on the vibrations of the S1 state, especially

in the low-frequency region up to several hundred wavenumbers. The amide I or

amide II region as well as NH stretching modes could not be investigated. Although

the work of Levy’s group lead to phantastic spectroscopic results, the main drawback

was that spectra could not be clearly interpreted: (1) it could not be excluded that the

spectra result from an overlay of different isomers, and (2) the computer power

available in the late 1980s made it impossible to get any reliable prediction of

vibrational spectra of different isomers. Furthermore, the structures of S1 states can

4 SPECTROSCOPY OF NEUTRAL PEPTIDES IN THE GAS PHASE

still not be predicted with an accuracy available for S0 states. Even S0 state

calculations on relative energies and vibrations of tripeptides with hundreds of

possible isomers are a challenge with respect to available computer resources.

Additionally, anharmonicities must be accounted for, especially for low-frequency

vibrations. All these problemsmade it nearly impossible to get a reliable interpretation

of the spectra obtained by Levy’s group. With the development of new spectroscopic

techniques (the double-resonance methods), different isomers could experimentally be

distinguished and vibrational frequencies in the amide I or II region as well as the NH

stretching modes could be recorded. The relevant techniques will be described in

Section 1.2. Additionally, the rapid increase of computer power since the 1990s has

made it possible to get reliable predictions of the structure of isolated large molecules

in the gas phase. As a consequence of the technical improvements, several

investigations on amino acid model systems, amino acids and peptides began in the

late 1990s and have become a rapidly growing field of scientific research, which will

be described in detail in Sections 1.3–1.6.

Although this chapter focuses mainly on different laser spectroscopic methods,

new developments within the field of microwave spectroscopy are also discussed,

yielding very high resolution spectra and thus precise geometric information.

In the following sections different techniques are reviewed and then their

applications starting with selected model systems up to the peptides are discussed.

An outlook on remaining issues is given at the end of the chapter.

1.2. EXPERIMENTAL SETUPS AND METHODS

1.2.1. Laser Spectroscopic Methods and Microwave Spectroscopy

Several spectroscopic techniques have been developed in order to analyze the

electronic ground and excited states of isolated biomolecules in the gas phase. Both

rotational resolution and vibrational spectra yield information on the structure of the

investigated systems. The fluorescence techniques offer some insight, especially in

the ‘‘low frequency’’ region of the S0 and S1 states of the investigated amino acids

and peptides (see Sections 1.4–1.6). In dispersed fluorescence (DF) spectroscopy

(see Figure 1.1a) the excitation laser frequency is fixed and the fluorescence light is

dispersed, yielding information on the S0 state. In the case of the laser-induced

fluorescence (LIF) technique, the excitation laser is scanned and the integral of the

complete fluorescence light is detected (Figure 1.1b). This method gives information

on the (low) vibrational frequencies of the electronically excited state. Another

method used to obtain frequencies of the S1 state is the resonant two-photon

ionization (R2PI) method (Figure 1.1c). As in LIF, the excitation laser is scanned,

but now a second photon from the same laser (one-color R2PI) or another UV laser

(two-color R2PI) is used to ionize the investigated species. Ions are observed

efficiently only when the first laser is in resonance with a vibrational (rotational)

level of the S1 state. If this vibronic level lives long enough with respect to the pulse

duration of the laser, the absorption of a second laser photon is enhanced, leading to

EXPERIMENTAL SETUPS AND METHODS 5

the production of ions. It should be mentioned that this chapter focuses mainly on

investigations with nanosecond (ns) laser systems.

A major advantage of the R2PI method compared with the LIF technique is the

possibility of combining spectroscopy with mass spectrometry; thus, ions produced by

the R2PI process can be mass-selected and one thus obtains direct information of the

investigated species (see Figure 1.2 and Section 1.2). In a molecular-beam experiment

containing mainly the monomer, usually other species are present, such as clusters of

the monomer or clusters with water, which can often not be removed completely from

the gas lines. Thus R2PI spectroscopy is a mass- and isomer-selective method.

The very important window of the NH stretching modes as well as the amide I

and II modes has been opened by the development of combined IR/UV techniques.

The combination of IR spectroscopy with both fluorescence (IR/LIF) and the R2PI

technique (IR/R2PI; see Figure 1.1d) are used. In both methods the UV laser photon

is fixed to one electronic transition that belongs to a selected isomer (originating

FIGURE 1.1. Different laser spectroscopic techniques applied to isolated amino acids or

peptides: (a) dispersed fluorescence, analyzing vibrations in the S0 state; (b) laser-induced

fluorescence to analyze vibrations in the S1 state; (c) resonant 2-photon ionization applied for

investigating vibrations of the excited state; (d, e) infrared/R2PI technique to investigate IR

spectra for the electronic ground and excited state, respectively; (f) UV/UV hole burning to

analyze different isomers appearing in one R2PI spectrum (for further details, see text).


from its vibrational ground state in the S0 state). By scanning an IR laser, different

vibrational modes in the S0 state can be excited. If the IR laser is in resonance with a

vibrational level of the S0 state, the vibrationless ground state is depopulated. By

firing the UV laser after the IR laser, the efficiency of the UV excitation is reduced,

since the number of molecules in the vibrationless ground state is reduced by the

resonant IR excitation. A reduction in UV laser excitation efficiency leads to a

decrease of the fluorescence signal (in the case of the IR/LIF technique) or to a

decrease of the R2PI signal (in the case of the IR/R2PI method). Thus both methods

indirectly yield an IR spectrum of a selected isomer in the S0 state by recording the

intensity of the LIF or R2PI signal as a function of the chosen IR wavelength. Like

the R2PI technique, the IR/R2PI method is also mass- and isomer-selective. This

method is also state-selective with respect to the different vibrational levels of the S0state. Historically, the first IR/R2PI spectrum was recorded by Page et al. (1988); a

plethora of publications have followed in this field (see Sections 1.3–1.6), beginning

with the investigations of Brutschy (Riehn et al. 1992), Mikami (Tanabe et al. 1993),

and Zwier (1996) and their coworkers. In these publications also the abbreviations

IR/UV double resonance, IR hole burning, and RIDIR spectroscopy are chosen

instead of IR/R2PI. The authors’ group published the first IR/R2PI spectrum in the

Time-of-flightmass spectrometer

Microchannelplates

Ionsignal

Digitaloscilloscope

Computerdata analysis

Ion lenses

y deflection

x deflection

Acceleration plates inWiley–McLaren arrangement

IR laser UV excitation laser

Molecules inhelium beam

Molecular beam

UV ionizing laser

Skimmer Pulsed valve

Driftregion

{FIGURE 1.2. Experimental setup to analyze the investigated species by R2PI, IR/R2PI, or

UV/UV hole-burning spectroscopy. The ions produced by two UV photons of a neutral

species are mass-analyzed in a linear time-of-flight spectrometer. The peptides are introduced

via a coexpansion with a rare gas (He or Ar) in a pulse valve. This valve can be heated, or a

laser desorption (ablation) source can be located in front of the valve (not shown).


C��O stretching region (Gerhards et al. 2002). This became possible due to thedevelopment of a new laser system in the nanosecond regime that produces narrow-

bandwidth IR light (better than 0.1 cm�1) with high energy (�1 mJ now from 4.7 to10 mm) (Gerhards et al. 2002; Gerhards 2004).

The region of C��O stretching vibrations is important since the amide I and amideII vibrations are significant for the description of peptide structures. Instead of the

laser described earlier (Gerhards 2004), a free-electron laser (Oepts et al. 1994)

is used in different applications on peptides (see Sections 1.3–1.5). This laser

system is very powerful (typically�50 mJ in one macropulse) and covers a region of�40–2200 cm�1 but has the drawback of a relative low spectral resolution(�15 cm�1 around 6 mm).

Another new developed laser system for the region of �6 mm (which would besuitable for the investigations of peptides) is described by Kuyanov et al. (2004).

Here the IR light is produced by stimulated backward Raman scattering in solid

para-hydrogen at 4 K pumped by a near-infrared OPO/OPA system. In contrast to

the generation of IR light by DFM (Gerhards 2004), the bandwidth is larger (0.4

instead of 0.1 cm�1) and the output energy strongly depends on the frequency,ranging from 1.7 mJ at 4.4 mm to 120 mJ at 8 mm (Kuyanov et al. 2004).

IR light in the region of the NH stretching vibrations (�3450 cm�1) or OHstretching modes (�3650 cm�1, important for the investigation of hydrated clusters)is usually generated by difference frequency mixing, an OPO/OPA process [see, e.g.,

Huisken et al. (1993)] or by a combination of DFM and OPA [e.g., see Unterberg

et al. (2000)]. Details on the laser are partly given in the references on applications of

IR/LIF and IR/R2PI spectroscopy.

Finally, it should be mentioned that the IR/R2PI (IR/LIF) method can also be

used to determine vibrational transitions in the electronically excited state

(Figure 1.1e). This method has been introduced by Ebata et al. (1996). By

applying the IR/R2PI technique for the S1 state, this state is excited by one UV

photon and then the depopulation of the S1 state via a subsequent IR excitation is

detected by the decrease of the R2PI signal caused by the ionization with a

second UV photon. (In the case of the IR/LIF method for the S1 state, no second

UV photon is necessary. Here the decrease of the fluorescence caused by the IR

excitation is determined.)

A further double-resonance technique used for the analysis of peptides is the UV/

UV hole-burning method (see Figure 1.1f ) first applied by Lippert and Colson

(1989) to the phenol(H2O) cluster. In contrast to the IR/R2PI technique, not an IR

laser but a UV laser is scanned, while a second UV laser (fired after the first scanning

UV laser) is fixed to one wavelength, such as the electronic origin of one isomer.

This method is used to determine whether different electronic transitions belong to

the same isomer, i.e. when the first (scanning) UV laser is in resonance with an

electronic transition that belongs to the isomer excited by the second laser, either the

fluorescence or ion signal caused by the second UV laser decreases, since excitations

of the first laser already removed parts of the molecules in the beam. In contrast to

the IR/UV technique, both laser photons of the UV/UV method lead to a

fluorescence or ion signal. Thus the signals resulting from first and second laser have


to be separated. The lasers are usually fired within 100–300 ns so that laser with foci

of�1–2 mm can still spatially overlap. For instance: If both the first and second lasercreate ions by a two photon absorption, either (1) the resolution of the spectrometer

must be good enough to separate the ions produced by the two lasers or (2) the ions

are separated by the use of fast high-voltage switches that accelerate the ions

produced by the first laser into the opposite direction.

The double-resonance techniques IR/R2PI and UV/UV hole burning lead to a

selection of isomers and identification of the different species by their IR spectra that

can be fully recorded in the range of all characteristic vibrational transitions.

Another technique that provides additional information on the dynamical behavior

of a flexible molecule is (infrared–population transfer spectroscopy (IR-PTS) (Dian

et al. 2002b) as well as the hole-filling method, both introduced by Zwier and

coworkers (Dian et al. 2002b, 2004b) (see Figure 1.3). By applying this method, the

FIGURE 1.3. (a) Three isomers of NATMA (Ac–Trp–NHMe) (see Section 1.4 for further

details); (b, c) schemes for IR population transfer (IR/PT) and hole-filling spectroscopy [21].

By exciting one isomer selectively with an IR photon, the two other isomers can be

populated. After a further collisional cooling within the expanding beam, the cold molecules

of B are analyzed via a UV probe laser. Because of a loss of population in B after IR

excitation, the fluorescence quantum yield caused by the UV probe laser is reduced (see

Figure 1.14). By scanning the UV laser, one can determine the distribution with respect to all

isomers (hole-filling spectroscopy). [Figure taken from Dian et al. (2002b).]


molecules in a molecular beam are excited twice. First the molecules are excited

directly behind the nozzle of a pulse valve, a region where collisional cooling still

takes place. If different conformers of a peptide molecule are in the beam, IR

excitation of one conformer (via, e.g. a NH stretching mode) may lead to formation

of another conformer. By further expansion in the jet, the remaining collisional

cooling can freeze out the new population induced by the IR excitation. The

population of different species can be recorded via the LIF spectroscopy (or, in

principle, also via the R2PI method). By comparing the LIF spectra obtained with or

without the first IR excitation, the change of the population is determined. The

difference between the IR-PTS and IR hole-filling methods is that in the former

procedure, the IR laser is scanned and the UV laser is fixed to a resonant transition of

one selected conformer; thus, the efficiency of forming a selected isomer with

respect to the IR excitation is determined. In the hole-filling method the UV laser is

scanned whereas the IR laser is fixed to one vibrational transition. Here the quantum

yield of forming different isomers after one selected IR excitation is obtained. These

new methods have been successfully applied to different species, including

protected amino acids (Dian et al. 2002b, 2004a,b); see Section 1.4.

All techniques mentioned so far need an aromatic chromophore. The infrared/

resonance energy transfer (IR/RET) method developed by Desfrancois and

coworkers (Lucas et al. 2004) offers the possibility of recording IR spectra of

species without such a chromophore. This technique is an extension of the RET

method, where a neutral molecule (with a large total dipole moment of

approximately >2D) collides with a Xe atom that has been excited to a high-lying Rydberg state. The collision of the excited Xe atom with the neutral species

induces a resonant electronic energy transfer leading to an anionic species. The

produced dipole-bound anions or even quadrupole-bound anions (DBAs or QBAs)

have an excess electron in a very diffuse orbital and should retain the structure of the

neutral parent. Since almost no internal energy is added, this very soft ionization

process occurs without fragmentation even for weakly bound clusters. Because of

the influence on the dipole moment of the formerly neutral molecule on ionization,

this technique is mass- and structure-sensitive. If the investigated molecule is

resonantly excited by an IR photon prior to the RET process, the stored energy can

break weak intermolecular bonds (in clusters) or lead to an autodetachment of the

DBA on ionization by RET. Thus the RETwith IR excitation of the neutral species is

lower compared to the RET obtained for a molecule in its vibrational ground state,

resulting in a depletion of the anion signal. This method describes a very useful

supplement to other techniques that require aromatic chromophores. With this

method the spectra of the water dimer and the formamide water complex (Lucas

et al. 2004) have been recorded and have shown very good agreement to earlier gas-

phase and matrix investigations. In a more recent publication the same group

examined formamide and its dimer in order to test the capability for monomers and

strongly bound cluster with binding energies higher than the excess electron-binding

energy (EBE) of the DBA (Lucas et al. 2005).

A classical method applied to determine structural parameter is microwave

spectroscopy. A significant process with respect to efficiency and analysis of


principles of mass spectrometry applied to … fileusing anion photoelectron spectroscopy in the gas...

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