ABSTRACT
SAMPSON, JASON SCOTT. Development and Characterization of an Atmospheric Pressure Ionization Source Matrix-Assisted Laser Desorption Electrospray Ionization Coupled to Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Analysis of Biological Macromolecules. (Under the direction of David Charles Muddiman).
The field of mass spectrometry has grown tremendously over the past few decades due in large part to the continued application to new and interesting areas of exploration. The advent of soft ionization sources such as electrospray ionization and matrix-assisted laser desorption/ionization has dramatically increased the applications of mass spectrometry; in particular, analysis of complex biological samples. Electrospray ionization has demonstrated the capability to generate multiply-charged ions which increases the amount of information garnered from each analysis. Matrix-assisted laser desorption/ionization has demonstrated complex sample analysis requiring minimal sample preparation, which results in high throughput. As a result of these findings, there has been tremendous growth in the development of new ionization source technology in recent years for reducing sample preparation required prior to analysis for high throughput sample analysis and the generation of multiply-charged ions.
Demonstrated herein is the development and characterization of an atmospheric pressure ionization source called matrix-assisted laser desorption electrospray ionization (MALDESI). MALDESI is a hybrid combination of MALDI and ESI which utilizes laser desorption with electrospray postionization for the generation
of multiply-charged ions. Multiply-charged ions are of particular importance when
using Fourier transform mass spectrometry, due to the increase in resolving power
and mass accuracy with increasing charge on the molecule. Positive identification of
biological macromolecules is demonstrated utilizing top-down characterization of intact polypeptides as well as high mass accuracy utilizing internal calibration. A newly designed highly robust and versatile atmospheric pressure ionization platform is designed and developed for high precision analysis (i.e., imaging) and described in detail.
Solid- and liquid state analysis of three out of the four classes of biological molecules (carbohydrates, proteins, lipids) is demonstrated using the high precision versatile ionization platform. Ultraviolet and infrared MALDESI is demonstrated at various wavelengths (UV, 337 nm and 349 nm and IR, 2.94 µm and 10.6 µm) with and without ESI postionization for the generation of multiply-charged ions. The liquid-state MALDESI ionization process is characterized which may be described as laser desorption from a macroscopic electrospray droplet. MALDESI direct analysis with minimal or no sample preparation is demonstrated and applications for high throughput analysis of complex samples (i.e., glycans) are described.
© Copyright 2009 by Jason Scott Sampson
All Rights Reserved
Development and Characterization of an Atmospheric Pressure Ionization Source Matrix-Assisted Laser Desorption Electrospray Ionization Coupled to Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Analysis of Biological Macromolecules
by Jason Scott Sampson
A dissertation submitted to the Graduate Faculty of North Carolina State University In partial fulfillment of the Requirements for the degree of Doctor of Philosophy
Chemistry
Raleigh, North Carolina
July 8, 2009
APPROVED BY:
______David C. Muddiman Edmond F. Bowden Professor, Chemistry Professor, Chemistry Committee Chair
______Kenneth W. Hanck Jorge A. Piedrahita Professor, Chemistry Professor, Molecular Biomedical Sciences DEDICATION
This dissertation is dedicated to my wife Starla and our son Jacob. Without your love and support this body of work would not have been possible.
ii BIOGRAPHY
Jason Scott Sampson was born in Greensboro, North Carolina to Ken and Carol
Sampson. He is from a large close knit family with six brothers and three sisters with fourteen nieces and nephews (and counting). He continues to enjoy the great outdoors including kayaking, hiking, biking and riding motorcycles. Upon completion of his Bachelor of Science in chemistry at the University of North Carolina at
Greensboro he and his family moved to Raleigh, NC where he attended graduate school at North Carolina State University to work on his PhD in Chemistry under the direction of Dr. David C. Muddiman.
iii TABLE OF CONTENTS
LIST OF TABLES ...... x
LIST OF FIGURES ...... xi
LIST OF PUBLICATIONS ...... xiii
ABSTRACTS FOR PRESENTATIONS ...... xiii
1. Introduction ...... 1
1.1 Ionization Sources ...... 1
1.1.1 Matrix-Assisted Laser Desorption/Ionization ...... 1
1.1.2 Electrospray Ionization ...... 3
1.1.3 Atmospheric Pressure Hybrid Ionization ...... 5
1.2 Fourier Transform Ion Cyclotron Resonance ...... 9
1.2.1 FT-ICR Mass Spectrometry ...... 9
1.2.2 Resolving Power ...... 10
1.2.3 Mass Measurement Accuracy ...... 11
1.2.4 Limit of Detection ...... 13
1.3 Synopsis of Completed Research ...... 14
1.4 References ...... 19
2. Generation and Detection of Multiply-Charged Peptides and Proteins by Matrix-
Assisted Laser Desorption Electrospray Ionization (MALDESI) Fourier Transform
Ion Cyclotron Resonance Mass Spectrometry...... 24
iv 2.1 Introduction ...... 24
2.2 Experimental ...... 27
2.2.1 Materials ...... 27
2.2.2 MALDESI FT-ICR Mass Spectrometer ...... 27
2.2.3 Control Experiments ...... 29
2.3 Results and Discussion ...... 30
2.3.1 MALDESI of Peptides and Proteins ...... 30
2.3.2 Evidence in Support of ESI Charging Mechanism in MALDESI .... 31
2.4 Conclusions ...... 33
2.5 References ...... 34
3. Direct Characterization of Intact Polypeptides by Matrix-Assisted Laser
Desorption Electrospray Ionization Quadrupole Fourier Transform Ion Cyclotron
Resonance Mass Spectrometry ...... 37
3.1 Introduction ...... 37
3.2 Experimental ...... 39
3.2.1 Materials ...... 39
3.2.2 MALDESI QFT-ICR Mass Spectrometer ...... 40
3.3 Results and Discussion ...... 42
3.3.1 High Mass Measurement Accuracy of Polypeptides using Internal
Calibration ...... 42
v 3.3.2 Direct Intact and Product Ion Analysis of Polypeptides by
MALDESI-QFT-ICR-MS ...... 44
3.3.3 Sensitivity Determination Using the MALDESI Source ...... 46
3.4 Conclusions ...... 48
3.5 References ...... 49
4. Construction of a Versatile High Precision Ambient Ionization Source for Direct
Analysis and Imaging ...... 52
4.1 Introduction ...... 52
4.2 Experimental ...... 53
4.2.1 Materials ...... 53
4.2.2 Profilometery Measurements ...... 54
4.2.3 MALDESI-LTQ-FT Mass Spectrometry ...... 55
4.3 Source Design ...... 56
4.3.1 MALDESI Source Design and Construction ...... 56
4.3.2 Desorption and Ionization Source Versatility ...... 64
4.4 Results and Discussion ...... 65
4.4.1 Explorer Laser Beam Characterization ...... 65
4.4.2 MALDESI-LTQ-FT-ICR of Peptides and Proteins ...... 66
4.4.3 Liquid Drop Sample Analysis ...... 68
4.5 Conclusions ...... 68
4.6 References ...... 70
vi 5. Development and Characterization of an Ionization Technique for Analysis of
Biological Macromolecules: Liquid Matrix-Assisted Laser Desorption
Electrospray Ionization ...... 74
5.1 Introduction ...... 74
5.2 Experimental ...... 78
5.2.1 Materials ...... 78
5.2.2 liq-MALDESI-LTQ and LTQ-FT Mass Spectrometry ...... 78
5.3 Results and Discussion ...... 80
5.3.1 liq-MALDESI-LTQ Liquid Sample Analysis ...... 80
5.3.2 liq-MALDESI Ion Abundance Versus Sample Target Potential ..... 83
5.3.3 Contact Angle Versus Sample Target Potential ...... 84
5.3.4 liq-MALDESI-LTQ-FT-ICR Analysis of Peptides and Proteins ...... 85
5.3.5 liq-MALDESI-LTQ-FT-ICR Electrochemical Ionization ...... 89
5.4 Conclusions ...... 90
5.5 References ...... 92
6. Intact and Top-Down Characterization of Biomolecules and Direct Analysis using
Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Coupled to FT-
ICR Mass Spectrometry ...... 95
6.1 Introduction ...... 95
6.2 Experimental ...... 98
6.2.1 Materials ...... 98
vii 6.2.2 IR- and UV-MALDESI Mass Spectrometry ...... 99
6.3 Results and Discussion ...... 100
6.3.1 Solid-State IR-MALDESI-FT-ICR ...... 100
6.3.2 Liquid-State IR-MALDESI-FT-ICR ...... 104
6.3.3 Intact and Top-Down Analysis using IR-MALDESI-FT-ICR ...... 106
6.3.4 Direct Analysis using UV- and IR-MALDESI ...... 107
6.3.5 Carbohydrate Analysis using IR-MALDESI ...... 109
6.4 Conclusions ...... 111
6.5 References ...... 112
7. Atmospheric Pressure Infrared (10.6 µm) Laser Desorption Electrospray
Ionization (IR-LDESI) Coupled to a LTQ-FT-ICR Mass Spectrometer ...... 117
7.1 Introduction ...... 117
7.2 Experimental ...... 120
7.2.1 Materials ...... 120
7.2.2 IR-LDESI Source and LTQ Mass Spectrometer ...... 120
7.3 Results and Discussion ...... 121
7.4 Conclusions ...... 124
APPENDIX ...... 129
APPENDIX A ...... 130
Supplemental Figures ...... 130
APPENDIX B ...... 133
viii Glossary ...... 133
ix LIST OF TABLES
Table 4.1 MALDESI parts list……………………………………………….………..57
Table 4.2 Description of parts fabricated in-house………………………….….….58
x LIST OF FIGURES
Figure 1.1 Schematic of MALDI process……………………………………………… 2
Figure 1.2 Schematic of electrospray process………………………….……………. 3
Figure 1.3 Theoretical resolving power in FT-ICR………………………..….………11
Figure 2.1 Front and side views of the MALDESI source…………………………..28
Figure 2.2 MALDESI and nanoESI FT-ICR ACS comparison……………...……...30
Figure 2.3 NanoESI and MALDESI charge state comparison……………………..32
Figure 3.1 Top and front detailed views of the MALDESI source…………….……40
Figure 3.2 Internal calibration using MALDESI………………………………………43
Figure 3.3 MALDESI product-ion spectrum of melittin…………………………….. 45
Figure 3.4 Natural and SIL angiotensin I limits of detection…………….………….46
Figure 4.1 New MALDESI versatile ionization platform……………………………. 55
Figure 4.2 MALDESI ionization source schematic side view……………….………61
Figure 4.3 MALDESI ionization source schematic top view……………….……….63
Figure 4.4 Laser spot size profilometery with gold QCM electrode………………. 65
Figure 4.5 MALDESI FT-ICR mass spectra of myoglobin and lysozyme…………67
Figure 5.1 Diagram of liquid sample droplet………………………………….………76
Figure 5.2 Liquid-state MALDESI with and without ESI postionization…………...81
Figure 5.3 Liquid-state MALDESI potential plots………………………….…………83
Figure 5.4 Liq-MALDESI FT-ICR mass spectra without ESI postionization.……..87
Figure 5.5 Electrochemical oxidation in liq-MALDESI………………….……………89
xi Figure 6.1 Schematic of solid-state IR-MALDESI………………….……………….101
Figure 6.2 Schematic of liquid-state IR-MALDESI…………………….……………104
Figure 6.3 Top-down sequencing of myoglobin using solid-state IR-MALDESI...106
Figure 6.4 UV- and IR-MALDESI direct analysis…………………………….……..107
Figure 6.5 Liquid-state IR-MALDESI of glycans………………...………...………..110
Figure 7.1 Schematic and liquid IR-LDESI mass spectra with ESI…….…...... ….122
Figure 7.2 Schematic and liquid IR-LDESI mass spectra without ESI…………...123
xii LIST OF PUBLICATIONS
The research in this dissertation has resulted in the following publications
Peer Reviewed Manuscripts
1. Sampson, J.S., Hawkridge, A.M., Muddiman, D.C., Generation and Detection of Multiply-Charged Peptides and Proteins by Matrix-Assisted Laser Desorption Electrospray Ionization (MALDESI) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, Journal of the American Society for Mass Spectrometry, 2006, 17 (12), 1712-1716. 2. Sampson, J.S., Hawkridge, A.M., Muddiman, D.C., Direct Characterization of Intact Polypeptides by Matrix-Assisted Laser Desorption Electrospray Ionization (MALDESI) Quadrupole Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, Rapid Communications in Mass Spectrometry, 2007, 21 (7), 1150-1154. 3. Sampson, J.S., Hawkridge, A.M., Muddiman, D.C., Development and Characterization of an Ionization Technique for Analysis of Biological Macromolecules: Liquid Matrix-Assisted Laser Desorption Electrospray Ionization, Analytical Chemistry, 2008, 80, 6773-6778. 4. Sampson, J.S., Hawkridge, A.M., Muddiman, D.C., Construction of a Versatile High Precision Ambient Ionization Source for Direct Analysis and Imaging, Journal of the American Society for Mass Spectrometry, 2008, 19, 1527-1534. 5. Sampson, J.S., Murray, K.K., Muddiman, D.C., Intact and Top-Down Characterization of Biomolecules and Direct Analysis using Infrared Matrix- Assisted Laser Desorption Electrospray Ionization Coupled to FT-ICR Mass Spectrometry, Journal of the American Society for Mass Spectrometry, 2009, 20, 4, 667-673. 6. Sampson, J.S., Muddiman, D.C., Atmospheric Pressure Infrared (10.6μm) Laser Desorption Electrospray Ionization (IR-LDESI) Coupled to a LTQ Fourier Transform Ion Cyclotron Resonance Mass Spectrometer, Rapid Communications in Mass Spectrometry, 2009, 23, 1989-1992.
ABSTRACTS FOR PRESENTATIONS
1. Oral Presentation: “Matrix-Assisted Laser Desorption Electrospray Ionization (MALDESI) FT-ICR-Mass Spectrometry for Direct Analysis of Biological Molecules” 58th Southeastern Regional Meeting of the American Chemical Society, Augusta, GA, 2006, Jason S. Sampson, Adam M. Hawkridge and David C. Muddiman.
xiii 2. Oral Presentation: “Direct Analysis of Biological Molecules by Matrix Assisted Laser Desorption Electrospray Ionization (MALDESI) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry” 121st North Carolina American Chemical Society Meeting, Durham, NC, 2007, Jason S. Sampson, Adam M. Hawkridge and David C. Muddiman. 3. Oral Presentation: “Development and Characterization of Matrix-Assisted Laser Desorption Electrospray Ionization (MALDESI) Coupled to FT-ICR-MS for the Direct Analysis of Biological Molecules” 59th Southeastern Regional Meeting of the American Chemical Society, Greenville, SC, 2007, Jason S. Sampson, Adam M. Hawkridge and David C. Muddiman. 4. Poster Presentation: “Ionization Mechanism Study and Characterization of Matrix-Assisted Laser Desorption Electrospray Ionization (MALDESI) Coupled to FT-ICR Mass Spectrometry” 55th American Society for Mass Spectrometry Conference, Indianapolis, IN, 2007, Jason S. Sampson, Adam M. Hawkridge, David C. Muddiman. 5. Poster Presentation: “Liquid Matrix-Assisted Laser Desorption Electrospray Ionization (liq-MALDESI) Coupled to FT-ICR Mass Spectrometry for the Analysis of Biological Molecules” 25th Triangle Chromatography Discussion Group Symposium, Raleigh, NC, 2008, Jason S. Sampson, Adam M. Hawkridge, David C. Muddiman. 6. Oral Presentation: “Development of a New Ionization Source Liquid Matrix- Assisted Laser Desorption Electrospray Ionization and Investigation of the MALDESI Ionization Mechanism” 56th American Society for Mass Spectrometry Conference, Denver, CO, 2008, Jason S. Sampson, R. Brent Dixon, Adam M. Hawkridge and David C. Muddiman. 7. Oral Presentation: “Applications of Solid- and Liquid-State Infrared Matrix- Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) for Analysis of Biological Macromolecules and Tissue Imaging” 237th American Chemical Society National Meeting, Salt Lake City, UT, 2009, Jason S. Sampson, Kermit K. Murray, R. Brent Dixon, Troy Ghashghaei and David C. Muddiman. 8. Poster Presentation: “Elucidation of the MALDESI Ionization Mechanism using Deuterated Solvents, Remote Analyte Sampling Transport and Ionization Relay Coupled with FT-ICR Mass Spectrometry” 57th American Society for Mass Spectrometry Conference, Philadelphia, PA, 2009, Jason S. Sampson, R. Brent Dixon, David C. Muddiman.
xiv CHAPTER 1
Introduction
1.1 Ionization Sources
1.1.1 Matrix-Assisted Laser Desorption/Ionization
Matrix-assisted laser desorption/ionization (MALDI)1, 2 is a soft ionization
technique which has proven highly useful for the analysis of biological molecules. In
MALDI, the analyte is prepared by mixing with or depositing the analyte onto the matrix prior to laser desorption. The matrix functions to isolate the analyte molecules and to absorb laser irradiation. The matrix is ablated from the surface taking with it the encapsulated analyte molecules thereby protecting the analyte molecules from destructive heating.
MALDI offers the advantages of ionization from complex samples with little sample preparation; however, the ions generated are primarily singly- and sometimes double-charged. Samples must be introduced into the high vacuum region of the mass spectrometer making the samples inaccessible during analysis and requiring special preparation for vacuum sensitive samples (i.e., tissues).3 In
atmospheric pressure (AP) MALDI the sample remains in the high pressure region
outside the mass spectrometer thereby eliminating the vacuum compatibility issues,
however at the cost of decreased sensitivity.4 The MALDI desorption and ionization
process is not well understood; however, in the “lucky survivor” theory proposed by
Karas et. al.,5 singly- and doubly-charged ions are the “lucky survivors” of rapid re-
1 neutralization reactions that take Laser Mass Analyzer place in the desorption plume
H+ above the surface following laser Uex (Extraction Grid) desorption. The desorption plume Matrix Cation (e.g., Na+) + Analyte Na is a heterogeneous mixture of analyte, matrix and [analyte +
matrix] clusters with charges
Figure 1.1 Schematic of MALDI process ranging from neutral to highly-
charged. Photoionization of matrix molecules results in the release of electrons and
their subsequent loss to the environment inducing an overall positive charge on the
desorption plume. The highly-charged species in the desorption plume are rapidly re-neutralized resulting in primarily singly-charged ions. A schematic of the MALDI process is shown in Figure 1.1.
MALDI has demonstrated a high tolerance for many interfering matrices
commonly found in biological samples (i.e. salts and surfactants). The typical MALDI
spectrum will often have both protonated and sodium adducted peaks (sodium may
originate from the analyte and matrix). The two channels of ionization increase the complexity of the mass spectra in addition to making quantitation more difficult. One solution to this problem is addition of sodium to the sample, thereby increasing the probability of ionization by sodium adduction and reducing or eliminating the protonated channel. In MALDI, the laser fluence, “energy per unit area incidence,” is
2 an important factor in the desorption and ionization process.6 There exists a fluence
threshold, below which ionization does not occur. Likewise there exists an upper
limit above which fragmentation is observed and an increase in ion abundance is not
observed for increased laser fluence. The high spatial resolution of the laser spot (5-
200 μm) also makes it an ideal platform for biological tissue imaging applications.7
MALDI is a pulsed ionization source which is highly compatible with pulsed mass analyzers such as time of flight (TOF) and FT-ICR. The pulsed nature of laser desorption and the static nature of samples allows for re-interrogation for in depth study and characterization.
1.1.2 Electrospray Ionization
Electrospray ionization (ESI)8 is one of the softest ionization techniques
employed for analysis of biological molecules. In electrospray ionization (ESI)
analyte is dissolved into metal 4e-
2H OO+ 4H+ 2 2 an electrically conducting + + + + + + + + + + +
+ + + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ +
+ +
+ +
+ +
+ +
+ + + +
+ +
+ + + + + + +
+ + +
+ + + + +
+ + + + +
+ + + + + + + + +
+ + + + + +
+ solution which is + + + +
++ + +
+ + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + +
+ + + + +
+ + + +
+ + + +
+ + + + + +
+ + + + + + + + + + + + + +
+ + + + + +
+ + + + +
+ + + +
+ + + + MS
+ + +
+ + +
+ + +
+ + + + + + + + +
+ + + + + + + + +
++ + + + + + + + + + + + + + + + + + + + + + + + Analyzer + + + + + + + + + + + pneumatically pumped + + + 2 kV + + 30 V through an emitter Figure 1.2 Schematic of electrospray ionization biased at high potential
for the generation of charged droplets and ultimately charged analyte ions. A
schematic of ESI is shown in Figure 1.2. The high potential of the ESI emitter drives
3 the oxidation of water and produces excess protons in the ESI solution9 as shown
below in Equation 1.1.
−+ 2 2 ++→ 44)()(2 eHgOlOH (1.1)
The liquid solution exits the emitter tip in a Taylor cone held by the surface tension of the liquid and induced by ion attraction to the counter-electrode (MS inlet capillary). Electrosprayed droplets are ejected from the Taylor cone once the charge density exceeds the surface tension. The resulting charged droplets, some
containing analyte, evaporate during their flight to the counter-electrode, causing a
decrease in droplet volume and surface area and an increase in the charge density
on the droplet surface. The droplets undergo coulombic explosions once the charge
density exceeds the Rayleigh limit (Equation 1.2), resulting in smaller and smaller
highly-charged droplets.
32 qr = 64 oγεπ r (1.2)
Where qr is the charge on the droplet, εo is the permittivity of free space, γ is
the surface tension and r is the radius of the droplet. Along the course of the
evaporation process the analyte molecules may escape the droplet when the
charges on the analyte allow it to overcome the surface tension of the droplet,
desorbing as a charged molecule. The hydrophobicity of the analyte determines
when the analyte will desorb from the ESI droplet which in turn governs the amount
of charge density available and the charge state it will attain.
4 High salt concentrations commonly found in biological samples are problematic for analysis by ESI, often requiring sample clean-up prior to analysis by high performance liquid chromatography (HPLC) to prevent ion suppression. The main advantages of ESI are high compatibility with liquid separations techniques such as HPLC and generation of multiply-charged ions which extends the mass range proportional to the charge on the ion (z) and are amenable to structural analysis using dissociation techniques such as collision induced dissociation (CID).10
1.1.3 Atmospheric Pressure Hybrid Ionization
The recent growth in ionization source development can be attributed to the renewed interest in the analysis of biological molecules brought about by soft ionization sources such as MALDI and ESI. The development of MALDI and ESI has dramatically enhanced the capabilities for mass spectrometric analysis of biological molecules due to high salt tolerance in MALDI resulting in minimal sample preparation for high throughput and the ability to generate highly useful sequence data from multiply-charged ions in ESI. The importance of MALDI and ESI was
immediately recognized by the scientific community and in 2002 resulted in the
award of the Nobel Prize in chemistry to Tanaka and Fenn for “their development of
soft desorption ionization methods for mass spectrometric analyses of biological
macromolecules.”11 However, the main disadvantages are that MALDI cannot
5 generate multiply-charged ions and extensive sample preparation is required for
ESI.
In an effort to resolve these issues a number of new ionization sources have
been introduced. In 1986 Lubman demonstrated a new concept, integrating two
ionization methods for improved ionization using 1) infrared laser desorbed neutral
molecules postionized using multi-photon ionization with a UV laser12 and 2) laser
desorbed neutral molecules ionized by chemical ionization via a radioactive 63Ni source.13 Later, in 1994, Hill and co-workers introduced secondary electrospray
ionization of neutrals (SESI)14 whereby neutral gas-phase analyte molecules were
ionized by interaction with the electrospray plume. This was the first report of
utilizing electrospray as a postionization technique. It is through these developments that we developed our working definition of a hybrid ionization source. A hybrid
ionization source is an ionization source which utilizes properties from two or more
existing ionization sources for which there exists a distinct benefit or attribute from
each source.
A number of hybrid ionization sources have been developed that primarily generate singly-charged ions including laser desorption atmospheric pressure chemical ionization (LD-APCI) originally introduced by lubman13 then revisited by
Harrison15 in 2002 for analysis of peptides utilizing corona discharge for post-
desorption ionization. Laser induced acoustic desorption (LIAD) originally introduced
by Lindner16 utilizes indirect laser desorption/ionization of electrospray deposited
6 analyte by irradiating the back side of a metal foil onto which the sample was
deposited and more recently by Kenttamaa17 in which the desorbed neutrals were postionized using chemical ionization. Direct analysis in real time (DART)18 and
atmospheric pressure solids analysis probe (ASAP),19 are among other ionization
techniques, have demonstrated the ability for direct analysis at atmospheric
pressure. However, the one major disadvantage is that these sources generate only
singly-charged ions.
In 2004, Cooks and coworkers developed an atmospheric pressure ionization source for the generation of multiply-charged ions called desorption electrospray
ionization (DESI).20 In DESI, electrosprayed solvent is accelerated by nitrogen gas
and directed onto an analyte bearing surface. The solvent forms a thin film on the
analyte surface from which analyte bearing droplets are desorbed by continuous
impact of high velocity droplets. The desorbed droplets then undergo an ESI-like
desorption and ionization process generating multiply-charged ions. The importance
of DESI was immediately discerned as the first technique for generating multiply-
charged ions without the extensive sample preparation required for ESI which
sparked the interest of mass spectrometrists around the globe and spurred the
development of new hybrid ionization sources.
Electrospray-assisted laser desorption ionization (ELDI)21 was developed by
Shiea and coworkers in 2005 and demonstrated ultraviolet (UV) laser desorption of proteins from samples without the addition of an organic matrix utilizing ESI
7 postionization for generating multiply-charged ions. Through our independent efforts
in 2005 we began to develop an ionization source called matrix-assisted laser
desorption electrospray ionization (MALDESI)22 whereby analyte is mixed with an
organic matrix for UV laser desorption followed by ESI postionization yielding
multiply-charged ions. MALDESI and ELDI were developed at approximately the
same time and although the two sources are in principle the same we determined
that a UV absorbing matrix was required for laser desorption and ionization under
our experimental conditions, hence ‘matrix-assisted’ is included in the name of our
source.
Recently, several atmospheric pressure infrared (IR) laser based
desorption/ionization techniques have emerged including laser ablation electrospray
ionization (LAESI),23 atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI),24 infrared laser ablation desorption electrospray ionization
(IR LADESI),25 infrared matrix-assisted laser desorption electrospray ionization (IR-
MALDESI)26 and infrared laser desorption electrospray ionization (IR-LDESI).27 The common theme of these infrared laser desorption techniques is the use of the infrared laser for desorption and the generation of multiply-charged ions. The main benefit of infrared laser desorption compared to UV laser desorption is that endogenous water can be utilized as matrix thereby effectively eliminating the need for sample preparation. This is particularly important for tissue imaging applications in which the application of matrix may result in molecular migration on the tissue.
8
1.2 Fourier Transform Ion Cyclotron Resonance
1.2.1 FT-ICR Mass Spectrometry
In FT-ICR mass spectrometry ions generated by the ionization source are guided by hexapole ion guides and enter the ICR cell located inside the bore of a superconducting magnet. The ions are trapped axially by an applied trapping potential and radially by the magnetic field. Once inside the magnetic field the ions are induced to move in cyclotron motion as a result of the Lorentz force28 as defined by Equation 1.3.
×= BqvF (1.3)
The Lorentz force (F) is a function of the charge on the ion (q), velocity of the ion (v) and the cross product of the magnetic field (B). The magnetic field serves to bend the trajectory of the ion into a circular radius when the ions are excited by an RF excitation voltage applied to the excitation plates. The excitation voltage induces coherence in ions of the same m/z and increases the radius of the orbiting ion cloud near the detection plates. The excitation voltage is turned off and the passing ions induce an image current on the detection plates which appears as a sinusoidal wave as a function of time, which is fast Fourier transformed and converted to m/z values using Equation 1.4.
qB ω = o (1.4) c m
9 Where ωc is the cyclotron frequency, q is the charge on the ion, Bo is the magnetic field strength and m is the mass of the molecule.
1.2.2 Resolving Power
The resolving power (RP) describes of the ability to distinguish between two species in a mass spectrum based solely on their mass and the difference between the two masses29 and is defined by Equation 1.5.
m RP = (1.5) (FWHM) Δm
For resolving power, where m is the mass of the ion and Δm is the width of the peak at ½ its maximum height, larger values are achieved for narrower peaks, indicating the ability to distinguish between species that are similar in mass. In FT-ICR the resolving power is dependent on the charge of the ion and is defined by Equation
1.6 28, 30 shown below. For singly charged ions, typically generated by MALDI, the resolving power is relatively low as compared to the resolving power achieved from multiply charged ions, typically generated by ESI.
7 TqB10 x 1.274 x TqB10 nacq'o RP = (1.6) (FWHM) m
The resolving power for a molecule, such as ubiquitin (mass = 8,600 Da) at a magnetic field strength (B0) of 9.4 Tesla and an acquisition time (Tacq’n) of 1 second is approximately 14,000 for the singly charged species and enables distinguishing between two species with a mass difference of 0.6 Da. A plot of the theoretical
10 resolving power versus 180000 RP ~ 140000 10+ 160000 the mass to charge 140000 RP ~ 28000 120000 ratio for ubiquitin is 2+ 100000 80000 RP ~ 14000 shown in Figure 1.3. 60000 1+ Resolving Power Resolving By adding a second 40000 20000 charge to the same 0 0 4000 8000 m/z molecule the resolving Figure 1.3 Theoretical resolving power in FT-ICR for ubiquitin with increasing charge power increases two
fold to 28,000, which allows distinguishing between two species that are 0.3 Da
apart. A ten fold increase is realized for the 10+ charge state yielding a resolving
power of 140,000, which allows distinguishing between two species that are only
0.06 Da apart. The higher resolving power achieved by multiply charged ions in FT-
ICR-MS enables two chemically similar species to be distinguished based solely on
their mass to charge ratio (m/z) ratio.
1.2.3 Mass Measurement Accuracy
Cyclotron frequency in FT-ICR is proportional to the charge (q) on the ion and
inversely proportional to the mass (m) of the ion as shown in Equation 1.4.
Assuming the mass of the ion and the magnetic field strength (Bo) remain constant
in an experiment, an increase in the charge on the ion will result in an increased
cyclotron frequency (ω). The increased cyclotron frequency has a positive impact on
11 the mass measurement accuracy (MMA) achieved in FT-ICR. The effects of space charge in FT-ICR cause variations in the cyclotron frequency; frequency shifts that result in decreasing mass measurement accuracy. For two ion packets of the same mass with different charge, the effective change in frequency due to space charge induced frequency shift for a highly charged ion (high cyclotron frequency) is much less than the effective frequency change for a lower charge state ion (lower cyclotron frequency). Therefore the mass accuracy is better for higher charge state ions when compared to ions of the same mass with a lower charge state.
Mass measurement accuracy in FT-ICR-MS is calculated using Equation 1.7 in parts per million (ppm).
mass − mass MMA(ppm) = observed ltheoretica ×106 (1.7) mass ltheoretica
The most common method for achieving good MMA in FTICR is external calibration,
typically achieving ~50 parts-per-million (ppm). In external calibration a calibrant ion
such as PPG 1000 is measured in the mass spectrometer and the known exact
masses are used to calibrate the instrument. The analyte of interest is then measured and the resulting mass spectrum is calibrated using the previously recorded external calibration file. External calibration often results in poor MMA as a result of systematic error caused by space charge effects inside the ICR cell. Space charge effects are the result of ion population differences inside the ICR cell during measurement of the calibrant and analyte ions, which influences the measured cyclotron frequency. As the number of ions and charges on the ions in the ICR cell
12 can vary significantly the space charge effects also vary significantly from scan to scan resulting in poor MMA. Several methods to counteract the variation in the ion population and therefore space charge effects have been developed such as automatic gain control (AGC)31 and several calibration laws32-34 that more accurately
account for ion population differences.
One simple method to counteract the variable space charge effects
experienced between the calibrant and analyte ions on mass measurement
accuracy is internal calibration.35 In internal calibration the calibrant and the analyte ions are introduced into and measured simultaneously in the ICR cell and therefore are subjected to the same space charge effects. Each spectrum is then calibrated using the known exact masses of the internal calibrant resulting in high mass measurement accuracy, routinely around 1 ppm.
1.2.4 Limit of Detection
Detection in FT-ICR is a result of measuring the cyclotron frequencies of the
ions in the ICR cell and the current induced by charged molecules passing near the
detection plates, called the image current. In modern ICR theory, 200 charges are
necessary to overcome the resistance in the detection circuit of the ICR cell and
induce a measurable current. The absolute minimum limit of detection for singly
charged ions would necessarily be 200 ions. If the charge on the ion was increased
to 10, the theoretical limit of detection becomes 20 ions. It becomes clear that by
13 increasing the charge on the analyte of interest (generating multiply charged ions), high resolving power and high mass measurement accuracy are easily obtained with low limits of detection.
1.3 Synopsis of Completed Research
The research described in this dissertation covers a wide range of methods implemented to develop and characterize a new atmospheric pressure ionization source coupled to Fourier transform ion cyclotron resonance mass spectrometry.
The basis of our pursuit of this technology is for the development of a robust, high stability and versatile ionization platform for proteomic analysis of biological macromolecules from complex samples. The ability to generate multiply-charged ions from complex biological samples at atmospheric pressure with minimal sample preparation are the key figures of merit for the development of MALDESI.
The concept of hybrid ionization has been previously established; however, with the exception of ESI and DESI no other ionization technology was available for generation of multiply-charged ions. In building upon the idea of two separate techniques for desorption and ionization and the use of ESI as a postionization method a prototype source was developed using an UV nitrogen laser (337 nm) with
ESI postionization. Chapter 2 describes the initial development of the MALDESI prototype for analysis of peptides and proteins.22 Multiply-charged ions were detected using high resolving power high mass accuracy FT-ICR mass
14 spectrometry. The MALDESI generated multiply-charged ions are characterized by
comparison with ions generated by ESI.
ESI is often used in proteomic analysis due to the ability to generate multiply- charged ions. Multiply-charged ions allow for enhanced top-down fragmentation efficiency, yields information rich sequencing data and are of particular importance in intact and top-down experiments utilizing FT-ICR mass spectrometry due to increased resolving power and mass accuracy with increased charge on the molecule. Chapter 3 details the application of MALDESI to high mass accuracy intact and top-down characterization of polypeptides and the determination of the limits of detection.36 The limits of detection are an important indicator of the value of
a particular method of ionization/detection. The limits of detection of the MALDESI
source were investigated using a stable isotope labeled (SIL) internal standard.
Chapter 4 details the construction of a highly versatile ionization platform for
atmospheric pressure ionization.37 This platform enable a variety of desorption and
ionization techniques to be implemented onto a single platform. For UV laser
desorption a new high repetition rate computer controlled Explorer laser system is
implemented. The physical measurement of the laser spot size and a description of the high precision computer controlled translation stage are detailed. This high
precision source is designed for application to imaging mass spectrometry. A
complete parts list is included in an effort to foster growth and development of
ionization source technology.
15 One problem commonly encountered with matrix-assisted laser desorption
techniques (e.g., MALDI, MALDESI) is poor shot-to-shot reproducibility. This is
generally the result of analyte + matrix co-crystallization inhomogeneity and results in what has been described as the “sweet spot” effect. A “sweet spot” is an area of
the dried analyte + matrix sample that yields high ion abundance as compared to
other areas of the sample which demonstrate low ion abundances. In MALDI,
significant effort in reducing the “sweet spot” effect has been expended and includes
a number of matrix application techniques (e.g., ESI deposition) and the use of a
liquid sample. Liquid samples are not compatible with vacuum MALDI applications
unless utilizing a highly viscous liquid matrix such as glycerol.2, 38, 39 Drawing from
the MALDI literature and a recent publication by Loo et. al.,40 whereby a liquid
droplet is utilized in their ELDI source for atmospheric pressure laser
desorption/ionization of a sample mixed with organic matrix; we have implemented a
liquid sample (analyte mixed with an organic matrix) with our MALDES source and
generated multiply-charged ions. Chapter 5 illustrates application of the liquid-state
MALDESI technique whereby laser ablation from a liquid sample is implemented for
the purpose of increasing shot-to-shot reproducibility.41 Ionization was performed
with and without ESI postionization; giving insight into the liquid-state MALDESI ionization mechanism. Electrochemical ionization was observed and characterized using a molecule known to be ionizable by electrochemical ionization using ESI,42 which also gives insight into the charging mechanism by demonstrating oxidation of
16 liquid-state samples on the sample target prior to laser desorption. This
demonstrates that the solvent is also readily ionizable by electrochemical ionization,
giving rise to excess protons which due to electrostatics would necessarily reside on
the surface of the droplet as has been demonstrated for ESI droplets.
UV laser desorption typically utilizes an organic matrix for many applications;
however, this requires a sample preparation step that may be eliminated by
implementing an IR laser.43-46 IR laser desorption may use the water of a sample as an endogenous matrix. Chapter 6 describes the implementation of an infrared laser
(2.94 μm) onto the versatile ionization platform for atmospheric pressure analysis.26
Solid- and liquid-state IR MALDESI is demonstrated for a variety of biological molecules. Intact and top-down characterization of myoglobin is demonstrated for positive protein identification based on protein fragment sequence coverage using high resolving power high mass accuracy FT-ICR mass spectrometry. IR MALDESI direct analysis of milk and egg yolk are demonstrated and compared with data collected using a UV laser (349 nm). Carbohydrate analysis is demonstrated for O- glycans cleaved from mucin with high mass measurement accuracy and MS/MS sequencing for positive identification. The softness of ionization is demonstrated in this experiment with no fragmentation of the O-glycan which has been observed in
MALDI analysis of O-glycans.
Chapter 7 details the implementation of a CO2 laser (IR, 10.6 µm) onto the
versatile atmospheric pressure ionization platform.27 Ionization is demonstrated
17 without the addition of matrix in a new technique called infrared laser desorption electrospray ionization (IR-LDESI). Proteins were observed as multiply-charged ions laser desorbed from liquid droplets with ESI postionization. However, the control experiments without ESI postionization did not reveal ion signal. Interestingly, cytochrome c was not observed with ESI postionization; however, protein signal was observed without ESI. This may be due to the increased number of basic sites on the molecule resulting in the desorption of highly charged ions that are repelled by the ESI plume.
18 1.4 References
1. Karas, M.; Hillenkamp, F., Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10000 Daltons. Analytical Chemistry 1988, 60, 2299-2301.
2. Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T., Protein and Polymer Analysis up to m/z 100 000 by Laser Ionization Time-of-Flight Mass Spectrometry. Rapid Communications in Mass Spectrometry 1988, 2, 151- 153.
3. Caprioli, R. M.; Farmer, T. B.; Gile, J., Molecular imaging of biological samples: Localization of peptides and proteins using MALDI-TOF MS. Analytical Chemistry 1997, 69, 4751-4760.
4. Laiko, V. V.; Baldwin, M. A.; Burlingame, A. L., Atmospheric pressure matrix assisted laser desorption/ionization mass spectrometry. Analytical Chemistry 2000, 72, 652-657.
5. Karas, M.; Gluckmann, M.; Schafer, J., Ionization in matrix-assisted laser desorption/ionization: singly charged molecular ions are the lucky survivors. Journal of Mass Spectrometry 2000, 35, 1-12.
6. Dreisewerd, K.; Schurenberg, M.; Karas, M.; Hillenkamp, F., Influence of the Laser Intensity and Spot Size on the Desorption of Molecules and Ions in Matrix-Assisted Laser-Desorption Ionization with a Uniform Beam Profile. International Journal of Mass Spectrometry and Ion Processes 1995, 141, 127-148.
7. Lee, C. C.; Chang, D. Y.; Jeng, J.; Shiea, J., Generating multiply charged protein ions via two-step electrospray ionization mass spectrometry. Journal of Mass Spectrometry 2002, 37, 115-117.
8. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., Electrospray Ionization for Mass-Spectrometry of Large Biomolecules. Science 1989, 246, 64-71.
9. Blades, A. T.; Ikonomou, M. G.; Kebarle, P., Mechanism of Electrospray Mass-Spectrometry - Electrospray as an Electrolysis Cell. Analytical Chemistry 1991, 63, 2109-2114.
10. Kelleher, N. L.; Lin, H. Y.; Valaskovic, G. A.; Aaserud, D. J.; Fridriksson, E. K.; McLafferty, F. W., Top down versus bottom up protein characterization by
19 tandem high-resolution mass spectrometry. Journal of the American Chemical Society 1999, 121, 806-812.
11. Fenn, J. B.; Tanaka, K., Nobel Prize in Chemistry. http://nobelprize.org/nobel_prizes/chemistry/laureates/2002/ 2002.
12. Tembreull, R.; Lubman, D. M., Pulsed Laser Desorption with Resonant 2- Photon Ionization Detection in Supersonic Beam Mass-Spectrometry. Analytical Chemistry 1986, 58, 1299-1303.
13. Kolaitis, L.; Lubman, D. M., Detection of Nonvolatile Species by Laser Desorption Atmospheric-Pressure Mass-Spectrometry. Analytical Chemistry 1986, 58, 2137-2142.
14. Chen, Y. H.; Hill, H. H.; Wittmer, D. P., Analytical Merit of Electrospray Ion Mobility Spectrometry as a Chromatographic Detector. J. Microcolumn Sep. 1994, 6, 515-524.
15. Coon, J. J.; Harrison, W. W., Laser desorption-atmospheric pressure chemical ionization mass spectrometry for the analysis of peptides from aqueous solutions. Analytical Chemistry 2002, 74, 5600-5605.
16. Lindner, B., On the Desorption of Electrosprayed Organic-Compounds from Supporting Metal Foils by Laser-Induced Pressure Waves. International Journal of Mass Spectrometry and Ion Processes 1991, 103, 203-218.
17. Crawford, K. E.; Campbell, J. L.; Fiddler, M. N.; Duan, P.; Qian, K.; Gorbaty, M. L.; Kenttamaa, H. I., Laser-induced acoustic desorption/Fourier transform ion cyclotron resonance mass spectrometry for petroleum distillate analysis. Analytical Chemistry 2005, 77, 7916-7923.
18. Cody, R. B.; Laramee, J. A.; Durst, H. D., Versatile new ion source for the analysis of materials in open air under ambient conditions. Analytical Chemistry 2005, 77, 2297-2302.
19. McEwen, C. N.; McKay, R. G.; Larsen, B. S., Analysis of solids, liquids, and biological tissues using solids probe introduction at atmospheric pressure on commercial LC/MS instruments. Analytical Chemistry 2005, 77, 7826-7831.
20. Cooks, R. G.; Ouyang, Z.; Takats, Z.; Wiseman, J. M., Ambient mass spectrometry. Science 2006, 311, 1566-1570.
21. Shiea, J.; Huang, M. Z.; Hsu, H. J.; Lee, C. Y.; Yuan, C. H.; Beech, I.; Sunner, J., Electrospray-assisted laser desorption/ionization mass spectrometry for
20 direct ambient analysis of solids. Rapid Communications in Mass Spectrometry 2005, 19, 3701-3704.
22. Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C., Generation and detection of multiply-charged peptides and proteins by matrix-assisted laser desorption electrospray ionization (MALDESI) Fourier transform ion cyclotron resonance mass spectrometry. Journal of the American Society for Mass Spectrometry 2006, 17, 1712-1716.
23. Nemes, P.; Vertes, A., Laser Ablation Electrospray Ionization for Atmospheric Pressure, in Vivo and Imaging Mass Spectrometry. Analytical Chemistry 2007, In Press.
24. Konig, S.; Kollas, O.; Dreisewerd, K., Generation of highly charged peptide and protein ions by atmospheric pressure matrix-assisted infrared laser desorption/ionization ion trap mass spectrometry. Analytical Chemistry 2007, 79, 5484-5488.
25. Rezenom, Y. H.; Dong, J.; Murray, K. K., Infrared laser-assisted desorption electrospray ionization mass spectrometry. Analyst 2008, 133, 226-232.
26. Sampson, J. S.; Murray, K. K.; Muddiman, D. C., Intact and Top-Down Characterization of Biomolecules and Direct Analysis Using Infrared Matrix- Assisted Laser Desorption Electrospray Ionization Coupled to FT-ICR, Mass Spectrometry. Journal of the American Society for Mass Spectrometry 2009, 20, 667-673.
27. Sampson, J. S.; Muddiman, D. C., Atmospheric Pressure Infrared (10.6μm) Laser Desorption Electrospray Ionization (IR-LDESI) Coupled to a LTQ Fourier Transform Ion Cyclotron Resonance Mass Spectrometer. Rapid Communications in Mass Spectrometry 2009, 23, 1989-1992.
28. Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S., Fourier transform ion cyclotron resonance mass spectrometry: A primer. Mass Spectrometry Reviews 1998, 17, 1-35.
29. Sommer, H.; Thomas, H. A.; Hipple, J. A., The Measurement of E/M by Cyclotron Resonance. Physical Review 1951, 82, 697-702.
30. Marshall, A. G.; Guan, S. H., Advantages of high magnetic field for Fourier transform ion cyclotron resonance mass spectrometry. Rapid Communications in Mass Spectrometry 1996, 10, 1819-1823.
21 31. Syka, J. E. P.; Marto, J. A.; Bai, D. L.; Horning, S.; Senko, M. W.; Schwartz, J. C.; Ueberheide, B.; Garcia, B.; Busby, S.; Muratore, T.; Shabanowitz, J.; Hunt, D. F., Novel linear quadrupole ion trap/FT mass spectrometer: Performance characterization and use in the comparative analysis of histone H3 post-translational modifications. Journal of Proteome Research 2004, 3, 621-626.
32. Easterling, M. L.; Mize, T. H.; Amster, I. J., Routine part-per-million mass accuracy for high-mass ions: Space-charge effects in MALDI FT-ICR. Analytical Chemistry 1999, 71, 624-632.
33. Muddiman, D. C.; Oberg, A. L., Statistical evaluation of internal and external mass calibration laws utilized in Fourier transform ion cyclotron resonance mass spectrometry. Analytical Chemistry 2005, 77, 2406-2414.
34. Williams, D. K.; Muddiman, D. C., Parts-per-billion mass measurement accuracy achieved through the combination of multiple linear regression and automatic gain control in a Fourier transform ion cyclotron resonance mass spectrometer. Analytical Chemistry 2007, 79, 5058-5063.
35. Flora, J. W.; Hannis, J. C.; Muddiman, D. C., High-mass accuracy of product ions produced by SORI-CID using a dual electrospray ionization source coupled with FTICR mass spectrometry. Analytical Chemistry 2001, 73, 1247- 1251.
36. Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C., Direct characterization of intact polypeptides by matrix-assisted laser desorption electrospray ionization quadrupole Fourier transform ion cyclotron resonance mass spectrometry. Rapid Communications in Mass Spectrometry 2007, 21, 1150- 1154.
37. Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C., Construction of a Versatile High Precision Ambient Ionization Source for Direct Analysis and Imaging. Journal of the American Society for Mass Spectrometry 2008, 19, 1527-1534.
38. Sunner, J.; Dratz, E.; Chen, Y. C., Graphite Surface Assisted Laser Desorption/Ionization Time-of-Flight Mass-Spectrometry of Peptides and Proteins from Liquid Solutions. Analytical Chemistry 1995, 67, 4335-4342.
39. Dale, M. J.; Knochenmuss, R.; Zenobi, R., Graphite/liquid mixed matrices for laser desorption/ionization mass spectrometry. Analytical Chemistry 1996, 68, 3321-3329.
22 40. Peng, I. X.; Shiea, J.; Loo, R. R. O.; Loo, J. A., Electrospray-assisted laser desorption/ionization and tandem mass spectrometry of peptides and proteins. Rapid Communications in Mass Spectrometry 2007, 21, 2541-2546.
41. Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C., Development and characterization of an ionization technique for analysis of biological macromolecules: Liquid matrix-assisted laser desorption electrospray ionization. Analytical Chemistry 2008, 80, 6773-6778.
42. Vanberkel, G. J.; Zhou, F. M., Characterization of an Electrospray Ion-Source as a Controlled-Current Electrolytic Cell. Analytical Chemistry 1995, 67, 2916- 2923.
43. Overberg, A.; Karas, M.; Bahr, U.; Kaufmann, R.; Hillenkamp, F., Matrix- Assisted Infrared-Laser (2.94-Mu-M) Desorption Ionization Mass- Spectrometry of Large Biomolecules. Rapid Communications in Mass Spectrometry 1990, 4, 293-296.
44. Berkenkamp, S.; Karas, M.; Hillenkamp, F., Ice as a matrix for IR-matrix- assisted laser desorption/ionization: Mass spectra from a protein single crystal. Proceedings of the National Academy of Sciences of the United States of America 1996, 93, 7003-7007.
45. Little, M. W.; Laboy, J.; Murray, K. K., Wavelength dependence of soft infrared laser desorption and ionization. J. Phys. Chem. C 2007, 111, 1412- 1416.
46. Menzel, C.; Dreisewerd, K.; Berkenkamp, S.; Hillenkamp, F., Mechanisms of energy deposition in infrared matrix-assisted laser desorption/ionization mass spectrometry. International Journal of Mass Spectrometry 2001, 207, 73-96.
23 Chapter 2
Generation and Detection of Multiply-Charged Peptides and Proteins by Matrix-Assisted Laser Desorption Electrospray Ionization (MALDESI) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
2.1 Introduction
The field of mass spectrometry has grown tremendously over the past century
due in large part to the continued development of more powerful ionization
techniques, mass analyzer technologies, and ion dissociation techniques. New
ionization techniques inherently increase the breadth of applications amenable to
mass spectrometry thereby driving the discovery, development, implementation and
refinement of mass analyzers and ion dissociation methods. The advent of
electrospray ionization (ESI)1 and matrix-assisted laser desorption ionization
(MALDI)2, 3 resulted in a paradigm shift in the biological sciences whereby intact
biomacromolecules could be ionized and detected by mass spectrometry.
The benefits of direct analysis of samples under ambient conditions with minimal or no sample pretreatment has lead to the development of a wide range of
novel ionization methods with potentially extraordinary impact in numerous fields
(e.g., environmental, forensics, material science, biomedical). These include fused-
droplet electrospray ionization (FD-ESI)4, laser desorption atmospheric pressure
chemical ionization (LD-APCI)5, desorption electrospray ionization (DESI)6, direct analysis in real time (DART)7, atmospheric-pressure solids analysis probe (ASAP)8,
24 and electrospray-assisted laser desorption/ionization (ELDI)9, 10. Interestingly,
modern high performance mass analyzers are largely based on hybrid technology
(e.g., coupling of linear ion trap with a Fourier transform mass spectrometer11) and similarly, so are these new ionization techniques.
Electrospray ionization is inherently well-suited for FT-ICR MS due to the inverse relationship between cyclotron frequency and m/z.12 Because cyclotron
frequency dictates mass resolving power, mass accuracy, limits of detection, and
top-down tandem MS efficiencies, the multiple charging (lower m/z) afforded by ESI
provides significant advantages.13 Matrix assisted laser desorption ionization
(MALDI) can also be coupled to FT-ICR MS where it can be a very powerful
technique for characterizing low molecular weight peptides and metabolites (<3-5
kDa). MALDI is a pulsed ionization technique and ESI is continuous; the ability to
analyze multiply-charged clinically-derived low abundant proteins benefits from the ability to interrogate them over long periods of time with multiple MS/MS techniques
(e.g., CID, SORI, IRMPD, ECD). However, because MALDI generates primarily
singly and doubly charged ions, the benefits of FT-ICR MS are not realized for high
molecular weight species. Thus, an ionization technique that combines the multiple-
charging of ESI and the pulsed nature of MALDI would be highly advantageous for
the top-down FT-ICR MS characterization of macromolecules and the analysis of
complex mixtures (i.e., high mass resolving power can be obtained without
consuming material).
25 Our specific interests in developing a new ionization source which retained the multiple-charging phenomena but did not require extensive sample preparation and/or on-line separations was for intact14 and top-down proteomics15, 16 as well as
the direct analysis of complex samples (e.g., low molecular weight fraction of
serum)17, 18. FT-ICR-MS is uniquely suited for these (and other) applications
because these applications demand high mass resolving power and mass accuracy.
Herein, we report the coupling of an ionization source dubbed matrix-assisted
laser desorption electrospray ionization (MALDESI) to an electrospray ionization
Fourier transform-ion cyclotron resonance mass spectrometer (ESI-FT-ICR-MS).
Our ionization source is in principle identical to the recently introduced ELDI source9,
10. However, unlike ELDI which does not require matrix, we found matrix was
necessary to obtain appreciable signal. Therefore, to differentiate between pure laser desorption and matrix assisted desorption, we have proposed the acronym
MALDESI. Furthermore, our results also support previous observations9, 10 that the detected ions were generated by the electrospray process rather than in the initial matrix-assisted laser desorption event (vide infra). However, if further experimentation reveals that a matrix is not required for a specific application, we fully support the use of the acronym ELDI. The determining factors, which dictate whether matrix is required (MALDESI) or not (ELDI) will likely be the limits of detection and molecular weight range accessible.
26 2.2 Experimental
2.2.1 Materials
Human bradykinin, angiotensin I, and glucagon as well as bovine ubiquitin,
2,5-dihydroxybenzoic acid (DHB), Fluka brand sinapinic acid (SA), and formic acid were purchased from Sigma-Aldrich. HPLC grade acetonitrile and high purity water were purchased from Burdick Jackson. All materials were used as received.
Electrospray solution was prepared by mixing acetonitrile and water (1:1 vol/vol) with 0.1% formic acid. The organic acid matrix was prepared by dissolving either 20 mg of SA or 150 mg DHB in 1 mL of the electrospray solution. The stainless steel sample target was spotted with a 1:1 (vol/vol) mixture of analyte (1 mg/mL) and matrix, covered and allowed to dry at ambient conditions. ESI experiments containing peptides and proteins were electrosprayed from 1 μM solutions.
2.2.2 MALDESI FT-ICR Mass Spectrometer
Figure 2.1 shows schematics of the MALDESI source and experimental
details. Figure 2.1A and 2.1B shows the side and front view, respectively, of our
overall experimental configuration of the ionization source. Figure 2.1C shows an
expanded side view of the source. In these experiments, an electrospray solution
was directly infused at 400 nL/min through a 75 μm i.d. fused silica capillary and 30
μm tapered PicoTip (New Objective, Woburn, MA) using a Harvard PHD-2000
27 syringe pump. The MALDESI source was custom built in-house and utilized a 337
nm pulsed nitrogen laser (Thermo Laser Science, VSL-337ND-S, Franklin, MA).
Two UV enhanced aluminum mirrors (part number 10D20AL.2) and a UV fused
silica convex lens with a 63 mm focal length (part number SPX017, Newport
Corporation, Irvine, CA) were used to direct and focus the laser beam, respectively.
The sample target plate was affixed to a XYZ linear stage (part number 460A,
Newport Corporation, Irvine, CA) for sample positioning.
Figure 2.1D shows the experimental pulse sequence. Mass spectra were acquired in positive-ion mode on a modified ESI-FT-ICR mass spectrometer
A C
Mirrors/Focusing Lens ESI Emitter FT-ICR MS Inlet FT-ICR MS (+1850 V) (+35 V) Capillary Inlet 5 mm ESI Emitter
Syringe Pump Matrix + Sample 3 mm ESI Source Insulator (Teflon™) XYZ Stage Sample Plate XYZ Stage (+550 V)
B Focusing Lens D
Mirrors FT-ICR MS 337 nm Laser Pulses Capillary Inlet 30 x 4 ns @ 120 μJ Detect UV Laser Excite (337 nm) XYZ Stage Accumulate Ion Injection/Trapping 45° 0 12345 Time (seconds)
Figure 2.1 A-C) front and side views of the MALDESI source. D) FT- ICR MS experimental pulse sequence.
28 (IonSpec FTMS Systems, Varian, Inc, Lake Forest, CA) with a 9.4 Tesla superconducting magnet (Cryomagnetics, Oak Ridge, TN). The laser was fired 30 times during a two-second period and the ions were accumulated in the external rf- only hexapole19 while the nanoESI source operated continually. The ions were then ejected from the hexapole, trapped in the ICR cell, excited and detected. All spectra reported are single-acquisition, collected with 512 k data points with a digitization rate of 1 MHz, a Blackman window function applied, and then zero-filled once prior to fast-Fourier transform.
2.2.3 Control Experiments
Throughout our investigations, control experiments were always conducted in order to verify the MALDESI process. These included experiments with the laser being fired without simultaneous ESI to rule out an atmospheric pressure MALDI
(AP-MALDI)20 as the ionization mechanism. Additionally, ESI was established without a laser event to rule out DESI6 as the ionization mechanism. These data are not shown, but in all cases, the absence of a signal ruled out AP-MALDI or DESI processes as the ionization mechanism. Furthermore, we did not observe signal without the use of an organic acid as a matrix, including analysis of tissue (data not shown), which is contrary to the ELDI reports9, 10 and may reflect specific source design and instrumental parameters.
29 2.3 Results and Discussion
2.3.1 MALDESI of Peptides and Proteins
Figure 2.2A and 2.2B show the single-acquisition MALDESI (top) and the
ESI (bottom) FT-ICR mass spectra of bradykinin and ubiquitin, respectively (see text in figure for details). The neutrals generated from the laser desorption events appear to be captured and charged by the nanoESI droplets. In Figure 2.2B, the
Single Acquisition MALDESI FT-ICR + 10+ [M + 10H ] Mass Spectrum of Ubiquitin ~8.6 kDa A 7.1 [M + 2H+]2+ B 1.5 * - Noise Peaks Sinapinic Acid, 30 Laser Shots ~10 Picomoles Ablated + 11+ [M + 11H ] Average Charge State = 9.58 Single Acquisition MALDESI FT-ICR + 9+ Mass Spectrum of Bradykinin ~1 kDa [M + 9H ] Sinapinic Acid, 30 Laser Shots [M + 12H+]12+ ~100 Picomoles Ablated [M + 8H+]8+ Average Charge State = 1.97 [M + 7H+]7+
[M + H+]1+ Absolute Abundance
Absolute Abundance * * 0 0
Single Acquisition nanoESI FT-ICR Mass Spectrum of Bradykinin ~1 kDa Single Acquisition nanoESI FT-ICR 2 Second Hexapole Accumulation Mass Spectrum of Ubiquitin ~8.6 kDa ~30 Femtomoles Injected 2 Second Hexapole Accumulation Average Charge State = 1.92 ~30 Femtomoles Injected Absolute Abundance
Absolute Abundance Average-Charge State = 9.73 42.4 500 600 700 800 900 1000 1100 22.9 m/z 700 800 900 m/z 1000 1100 1200 C 10 Ubiquitin (~8.6 kDa) D Approximate Efficiencies of Capturing Neutral Species Ablated 8 by Nitrogen Laser Co-Crystallized with Sinapinic Acid Matrix Angiotensin I m = 0.98 6 (~1.3 kDa) R2 = 0.9998 Peptide Bradykinin Angiotensin I Glucagon Ubiquitin 4 Glucagon Analyte:Matrix 1:100 1:100 1:300 1:800 MALDESI (~3.5 kDa) Neutral 2 Bradykinin (1 kDa) Capture 0.006% 0.006% 0.002% 0.02% Average Charge-State Charge-State Average 0 Efficiency 02 46810 Average Charge-State ESI
Figure 2.2 A-B) MALDESI (top) and nanoESI (bottom with inverted y-axis) FT-ICR mass spectra of bradykinin and ubiquitin, respectively. C) Plot of the average charge-state observed for bradykinin, angiotensin I, glucagon, and ubiquitin via nanoESI (x-axis) and MALDESI (y-axis). D) Approximate neutral capture efficiencies of the MALDESI source relative to nanoESI of the same peptides.
30 inset shows an expansion of the 10+ charge-state of ubiquitin demonstrating isotopic
resolution was achieved. It should be noted that the amount of material ablated from
the surface is a very conservative estimate and we believe it is significantly less.
Figure 2.2C shows the average charge-state observed by ESI (x-axis) and by
MALDESI (y-axis) for bradykinin (Figure 2.2A), angiotensin I (data not shown),
glucagon (data not shown) and ubiquitin (Figure 2.2B). The data is highly correlated
(slope = 0.98, R2 = 0.9998) providing strong evidence that ionization is occurring via
the ESI mechanism.
Figure 2.2D presents approximate neutral capture efficiencies based on the
amount of material spotted, size of the spot, laser spot size, number of laser shots
and the absolute abundance of each peptide or protein relative to their
corresponding nanoESI spectra. The neutral capture efficiencies range from 0.002 –
0.02% but we fully expect to be able to improve this with a capture cell or the use of an air amplifier.21-23 This is an active area of our research where we will utilize
natural and stable-isotope labeled peptides to accurately quantify overall capture
and ionization efficiencies of the MALDESI process.
2.3.2 Evidence in Support of ESI Charging Mechanism in MALDESI
In order to elucidate the role of ESI charging in the MALDESI process, we
performed three experiments. First, an equimolar mixture of both angiotensin I and
bradykinin were electrosprayed with no laser event (Figure 2.3A). Second,
31 ABAngiotensin I:Bradykinin FT-ICR MS FT-ICR MS C FT-ICR MS 1 μM Bradykinin (1 μM:1 μM) Inlet 1 μM Angiotensin I Inlet Inlet
No Sample DHB DHB + + Bradykinin Angiotensin I 5.9 23.6 4.6 [Bradykinin + 2H+]2+ [Bradykinin + 2H+]2+ [Bradykinin + 2H+]2+ ~100 picomoles ~30 fmoles Equilmolar Mixture ~30 femtomoles of each injected [Angiotensin I + 2H+]2+ ~30 femtomoles [Angiotensin I + 2H+]2+ + 2+ [Angiotensin I + 2H ] ~100 pmoles *DHB-Bradykinin adducts
+ 1+ [Bradykinin + H+]1+ Absolute Abundance Absolute Abundance Absolute * [Bradykinin + H ] Absolute Abundance Absolute * * 500 600 700 800 900 1000 1100 500 600 700 800 900 1000 1100 500 600 700 800 900 1000 1100 m/z m/z m/z Figure 2.3 A) nanoESI FT-ICR mass spectrum of an equimolar mixture of angiotensin I and bradykinin. B) MALDESI of bradykinin and simultaneous nanoESI of angiotensin I. C) MALDESI of angiotensin I and simultaneous nanoESI of bradykinin.
bradykinin was desorbed solely via the MALDESI process while angiotensin I was
produced via conventional ESI (Figure 2.3B). Third, the angiotensin I was desorbed
solely via the MALDESI process while bradykinin was produced via conventional ESI
(Figure 2.3C). Figure 2.3A shows the nanoESI FT-ICR mass spectrum of an equimolar mixture of angiotensin I and bradykinin. The [M+2H+]2+ in Figure 2.3A
was observed to be the dominant charge-state as in Figure 2.2C with an abundance
ratio of about 4:1 favoring bradykinin. Interestingly, Figure 2.3B shows that even though the bradykinin is produced by the 30 laser shots from the surface and subsequently captured and charged by the ESI droplets, bradykinin remains more abundant than the electrosprayed angiotensin I at 1 μM. Conversely, in Figure 2.3C the MALDESI-generated bradykinin remains the dominate species while the electrosprayed angiotensin I is barely observed. This is consistent with the ESI
32 mechanism observed in Figure 2.3A where bradykinin out competes the angiotensin
I by a factor of 4 in the electrospray process. Thus, based on this observation, one
would expect angiotensin I to produce a low abundance signal given the
combination of charge-competition with bradykinin and the lower neutral
capture/ionization efficiency of the MALDESI process (Figure 2.2D).
2.4 Conclusions
It is clear that we must better understand the mechanisms of MALDESI before we can confidently apply it to solve real biological problems. Given that
MALDESI is a hybrid of MALDI, ESI, and ELDI, there is an enormous amount of experimental space for which to research. We are currently in the process of using stable isotope labeled internal standards to probe the mechanism of MALDESI and determine the limits of detection of our source. Furthermore, we are actively pursuing the role in which the ESI solvent composition, types of organic acid matrix, and matrix crystal morphology, play in the generation of MALDESI ions. Finally, we are working on improving sensitivity of the method (e.g., neutral capture efficiency, ion transmission) including the implementation of the air amplifier. These lines of research will result in an optimized and reasonably understood ionization source, which will enable us to commence applications including intact and top-down proteomic analysis using a Q-FTICR as well as direct analysis of biofluids and tissue.
33 2.5 References
1. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., Electrospray Ionization for Mass-Spectrometry of Large Biomolecules. Science 1989, 246, 64-71.
2. Karas, M.; Hillenkamp, F., Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10000 Daltons. Analytical Chemistry 1988, 60, 2299-2301.
3. Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T., Protein and Polymer Analysis up to m/z 100 000 by Laser Ionization Time-of-flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 1988, 2, 151-153.
4. Lee, C. C.; Chang, D. Y.; Jeng, J.; Shiea, J., Generating multiply charged protein ions via two-step electrospray ionization mass spectrometry. J Mass Spectrom 2002, 37, 115-117.
5. Coon, J. J.; Harrison, W. W., Laser desorption-atmospheric pressure chemical ionization mass spectrometry for the analysis of peptides from aqueous solutions. Analytical Chemistry 2002, 74, 5600-5605.
6. Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G., Mass spectrometry sampling under ambient conditions with desorption electrospray ionization. Science 2004, 306, 471-473.
7. Cody, R. B.; Laramee, J. A.; Durst, H. D., Versatile new ion source for the analysis of materials in open air under ambient conditions. Analytical Chemistry 2005, 77, 2297-2302.
8. McEwen, C. N.; McKay, R. G.; Larsen, B. S., Analysis of solids, liquids, and biological tissues using solids probe introduction at atmospheric pressure on commercial LC/MS instruments. Analytical Chemistry 2005, 77, 7826-7831.
9. Huang, M. Z.; Hsu, H. J.; Lee, J. Y.; Jeng, J.; Shiea, J., Direct Protein Detection from Biological Media through Electrospray-Assisted Laser Desorption Ionization/Mass Spectrometry. Journal of Proteome Research 2006, 5, 1107-1116.
10. Shiea, J.; Huang, M. Z.; Hsu, H. J.; Lee, C. Y.; Yuan, C. H.; Beech, I.; Sunner, J., Electrospray-assisted laser desorption/ionization mass spectrometry for direct ambient analysis of solids. Rapid Communications in Mass Spectrometry 2005, 19, 3701-3704.
34 11. Syka, J. E.; Marto, J. A.; Bai, D. L.; Horning, S.; Senko, M. W.; Schwartz, J. C.; Ueberheide, B.; Garcia, B.; Busby, S.; Muratore, T.; Shabanowitz, J.; Hunt, D. F., Novel linear quadrupole ion trap/FT mass spectrometer: performance characterization and use in the comparative analysis of histone H3 post-translational modifications. J Proteome Res 2004, 3, 621-626.
12. Henry, K. D.; Williams, E. R.; Wang, B. H.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. F., Fourier-Transform Mass Spectrometry of Large Molecules by Electrospray Ionization. Proc. Natl. Acad. Sci. USA 1989, 86, 9075-9078.
13. Marshall, A. G.; Guan, S. H., Advantages of high magnetic field for Fourier transform ion cyclotron resonance mass spectrometry. Rapid Commun. Mass Spectrom. 1996, 10, 1819-1823.
14. Nepomuceno, A. I.; Mason, C. J.; Muddiman, D. C.; Bergen, H. R., 3rd; Zeldenrust, S. R., Detection of genetic variants of transthyretin by liquid chromatography-dual electrospray ionization fourier-transform ion-cyclotron- resonance mass spectrometry. Clin Chem 2004, 50, 1535-1543.
15. Kelleher, N. L., Top-down proteomics. Anal Chem 2004, 76, 197A-203A.
16. Reid, G. E.; McLuckey, S. A., 'Top down' protein characterization via tandem mass spectrometry. J Mass Spectrom 2002, 37, 663-675.
17. Bergen, H. R., 3rd; Vasmatzis, G.; Cliby, W. A.; Johnson, K. L.; Oberg, A. L.; Muddiman, D. C., Discovery of ovarian cancer biomarkers in serum using NanoLC electrospray ionization TOF and FT-ICR mass spectrometry. Dis Markers 2003, 19, 239-249.
18. Johnson, K. L.; Mason, C. J.; Muddiman, D. C.; Eckel, J. E., Analysis of the low molecular weight fraction of serum by LC-dual ESI-FT-ICR mass spectrometry: precision of retention time, mass, and ion abundance. Anal Chem 2004, 76, 5097-5103.
19. Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D.-H.; Marshall, A. G., External Accumulation of Ions for Enhanced Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. J. Am. Soc. Mass Spectrom. 1997, 8, 970-976.
20. Laiko, V. V.; Baldwin, M. A.; Burlingame, A. L., Atmospheric pressure matrix- assisted laser desorption/ionization mass spectrometry. Anal Chem 2000, 72, 652-657.
35 21. Hawkridge, A. M.; Zhou, L.; Lee, M. L.; Muddiman, D. C., Analytical performance of a venturi device integrated into an electrospray ionization fourier transform ion cyclotron resonance mass spectrometer for analysis of nucleic acids. Anal Chem 2004, 76, 4118-4122.
22. Yang, P.; Cooks, R. G.; Ouyang, Z.; Hawkridge, A. M.; Muddiman, D. C., Gentle protein ionization assisted by high-velocity gas flow. Anal Chem 2005, 77, 6174-6183.
23. Zhou, L.; Yue, B.; Dearden, D. V.; Lee, E. D.; Rockwood, A. L.; Lee, M. L., Incorporation of a venturi device in electrospray ionization. Anal Chem 2003, 75, 5978-5983.
36 Chapter 3
Direct Characterization of Intact Polypeptides by Matrix-Assisted Laser Desorption Electrospray Ionization Quadrupole Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
3.1 Introduction
Mass spectrometry has become one of the most valuable technologies for the identification and characterization of biological molecules. This growth has been driven in large part by the introduction and development of soft ionization techniques such as electrospray ionization (ESI)1 and matrix-assisted laser desorption ionization
(MALDI).2, 3 Following the development of ESI and MALDI, researchers began to
develop hybrid ionization techniques that were designed to minimize sample
preparation and exploit the benefits of ambient mass spectrometric analysis.
Ambient ionization sources such as fused-droplet electrospray ionization (FD-ESI)4,
laser desorption atmospheric pressure chemical ionization (LD-APCI)5, desorption
electrospray ionization (DESI)6, direct analysis in real time (DART)7, atmospheric-
pressure solids analysis probe (ASAP)8 and electrospray-assisted laser desorption/ionization (ELDI)9, 10 combine these desirable attributes and have
furthered the potential applications of mass spectrometry. The development of
these and other new ionization sources continue to augment the overall applicability
of mass spectrometry to solve increasingly complex biological problems.
Modern Fourier transform ion cyclotron resonance (FT-ICR) mass
spectrometers used for bioanalytical and proteomics experiments typically operate in
37 an optimal mass-to-charge (m/z) range of between 300 and 3000 m/z. This is due to
the inverse relationship between cyclotron frequency and m/z11 and the
“optimization” of several analytical figures of merit at higher cyclotron frequencies
(e.g., resolving power, mass measurement accuracy, MS/MS dissociation
efficiency).12 In order to access these unparalleled figures of merit, large biological
macromolecules must be multiply-charged. Thus, ESI is much more widely used in
FT-ICR MS than MALDI which generates primarily singly charged ions.
Although ESI generates multiply-charged ions amenable to FT-ICR MS analysis, ESI is a continuous ionization source and thus, many of the ions generated are never measured when coupled to a pulsed mass analyzer such as a FT-ICR MS.
In contrast, MALDI is a pulsed ionization source which is more compatible with FT-
ICR. Recently, we introduced matrix-assisted laser desorption electrospray ionization (MALDESI) which is a hybrid ionization source that combines the desirable attributes of ESI (e.g., generation of multiply charged ions) and MALDI
(e.g., extended interrogation of samples with minimal preparation) into an integrated pulsed ionization source that generates multiply-charged ions.13 The characterization
of this new ionization source is important to demonstrate the inherent capabilities as
well as reveal potential limitations of the MALDESI source.
Herein, we report the direct detection of a peptide with 1 part-per-million mass
accuracy and its top-down characterization using hybrid FT-ICR technology.
Furthermore, we demonstrate sub-femtomole sensitivity.
38 3.2 Experimental
3.2.1 Materials
Melittin, angiotensin I, 2,5-dihydroxybenzoic acid (DHB), and formic acid were purchased from Sigma-Aldrich. The stable isotope labeled (SIL) version of
13 15 angiotensin I ( C5 N substituted valine, MW 1302) was purchased from ISOTEC
(Sigma-Aldrich). HPLC grade acetonitrile and high purity water were purchased from Burdick Jackson. The iodopeptides were designed to have a sufficient mass excess to avoid overlap with endogenous peptides and proteins14 and were
synthesized in the Mayo Proteomics Research Center (Mayo Clinic, Rochester, MN).
These four iodopeptides (I1-I4) had the sequences (I2Y)GK (I1), (I2Y)GK(I2Y)G (I2),
(I2Y)SR(I2Y)GSYGSSI (I3), and (I2Y)SR(I2Y)GSYGSSIGSY (I4) with neutral
monoisotopic masses of 617.9836, 1089.8617, 1742.1433, and 2049.2601 Da
14 respectively; (I2Y) is 3,5-diiodo-tyrosine. All materials were used as received.
The electrospray solution was prepared by mixing acetonitrile and water (1:1 vol/vol) with 0.1% formic acid. The organic acid matrix was prepared by dissolving
150 mg DHB in 1 mL of the electrospray solution. The stainless steel sample target was spotted with a 1:1 (vol/vol) mixture of analyte and matrix, covered and allowed to dry at ambient conditions. Iodopeptides and stable isotope labeled angiotensin I were electrosprayed from 1.35 and 1 µM (total concentration) solutions respectively.
The mixture of iodopeptides had the following concentrations, I1, I3 and I4 = 0.40
µM and I2 = 0.15 µM.
39 3.2.2 MALDESI QFT-ICR Mass Spectrometer
Figure 3.1 shows schematics of the MALDESI source and experimental
details. Figure 3.1A and 3.1B show the top and side views, respectively, of the
overall configuration of our ionization source coupled to a Z-Spray source. In these experiments, an electrospray solution was directly infused at 400 nL/min through a
75 μm i.d. fused silica capillary and 30 m tapered PicoTip (New Objective, Woburn,
MA) using a Harvard PHD-2000 syringe pump. The MALDESI source was custom
A built in-house FT-ICR MS Inlet 5 mm ESI Emitter and utilized a
Matrix + Sample 337 nm pulsed
nitrogen laser Focusing Lens UV laser B 337 nm FT-ICR MS Inlet with 120 μJ (+40 V)
ESI Emitter pulse energy (+2400 V) 5 mm and 4 ns pulse 45° 3 mm width (Thermo Sample Plate XYZ Stage (+550 V) Matrix + Sample Laser Science,
Figure 3.1. A-B) Top and front detailed views of the MALDESI source VSL-337ND-S, showing the relative positions and distances of the ESI emitter and sample plate to the mass spectrometer inlet and the relative orientation Franklin, MA). of the laser beam path. Two UV enhanced aluminum mirrors (part number 10D20AL.2) and a UV fused silica convex lens with a 63 mm focal length (part number SPX017, Newport
Corporation, Irvine, CA) were used to direct and focus the laser beam, respectively
40 to a spot size of approximately 200 µm in diameter. The ESI emitter was affixed to a
XYZ linear stage (part number 460A, Newport Corporation, Irvine, CA) for emitter tip positioning. The sample target plate was affixed to a custom made mounting bracket with an integrated XYZ linear stage for sample positioning.
Mass spectra were acquired in positive-ion mode on a hybrid ESI-QFT-ICR mass spectrometer (IonSpec FTMS Systems, Varian, Inc., Lake Forest, CA) equipped with a Z-spray ionization source (Waters/Micromass) and a 7.0 Tesla superconducting magnet (Cryomagnetics, Oak Ridge, TN). The nanoESI source operated continually. The laser was manually triggered to pulse 30 times during a two-second period and the ions were accumulated in the final hexapole in radio frequency-only mode for all ion collections15, except when performing tandem mass spectrometry (MS/MS). The ions were then ejected, trapped in the ICR cell, excited and detected. For MS/MS the precursor ions were isolated using the mass-selective quadrupole, collected and fragmented in the final hexapole before being ejected to the ICR cell for excitation and detection. All spectra reported are single-acquisition, collected with 1024 k data points with a digitization rate of 1 MHz, a Blackman window function applied, and then zero-filled once prior to fast-Fourier transform.
41 3.3 Results and Discussion
3.3.1 High Mass Measurement Accuracy of Polypeptides using Internal Calibration
MALDESI-QFT-ICR MS has the unique potential for simultaneously introducing analyte polypeptide ions prepared in a MALDI matrix and internal calibrant ions introduced via ESI. The internal calibrant species used in this study were iodinated peptides (Figure 3.2A). A 1.35 μM ESI solution (50:50 ACN:H2O
w/0.1% formic acid) was continuously electrosprayed while the UV laser was pulsed to induce matrix assisted laser desorption of melittin from the stainless steel MALDI plate (Figure 3.2A). Figure 3.2B shows the resultant MALDESI-QFT-ICR mass spectrum of melittin (analyte) and the electrosprayed four iodopeptides (internal calibrants). The I1 component of the mixture demonstrated a much lower abundance in the mass spectra as a result of having only one diiodo-tyrosine tag compared with two tags present in each of the other components, resulting in a lower hydrophobicity for I1 and therefore a reduced abundance when electrosprayed.
The accurate mass values are dependent on the ability of the instrument to
measure the cyclotron frequency inside the ICR cell.16 By simultaneously populating
the ICR cell with the internal calibrant and analyte, both the calibrant and analyte
experience the same space charge effects and variations in the trapping and
excitation potentials.16 The most accurate mass measurements were obtained when
the internal calibrants spanned the m/z range of the analyte.17 The mass
measurement accuracy (mean + 95 % confidence interval) for [melittin + 3H+]3+ and
42 QFT-ICR-MS A 1.35 μM Iodopeptides I1 Y G K I2 Y G K Y G Inlet
I3 Y S R Y G S Y G S S I Melittin + I4 Y S R Y G S Y G S S I G S Y DHB
Y = 3,5-diiodo-tyrosine + 2+ [I3+2H+]2+ [I4+2H ] B 13
[Melittin+4H+]4+ [I2+1H+]1+
[Melittin+3H+]3+ Arb. units Absolute Abundance [I1+1H+]1+
0 600 700 800 900 1000 1100 m/z
C Average MMA (mean + 95 % CI) ppm External calibration Internal calibration Fold Improvement [Melittin+3H+]3+ 10.6 (+ 2.1) -1.1 (+ 0.3) 9 [Melittin+4H+]4+ 4.5 (+ 1.5) -1.4 (+ 0.1) 3
Figure 3.2. A) Amino acid sequences of the 4 iodopeptides used for internal calibration. B) MALDESI QFT-ICR mass spectrum of melittin (1:1 in DHB) and the 4 iodopeptide (1.35 µM total concentration) mixture doped into the ESI solution. C) Mass measurement accuracy (mean + 95%CI) using external and internal calibration for 3+ and 4+ charge states of melittin.
[melittin + 4H+]4+ were determined before and after internal calibration and the results are shown in the table in Figure 3.2C. The average mass measurement accuracies using external calibration parameters were 10.6 (+ 2.1) ppm ([melittin +
3H+]3+) and 4.5 (+ 1.5) ppm ([melittin + 4H+]4+). The results following internal
43 calibration were found to be -1.1 (+ 0.3) ppm and -1.4 (+ 0.1) ppm respectively, resulting in greater than 9-fold and 3-fold improvement in mass measurement accuracy. These results are equivalent to the mass measurement accuracy we recently demonstrated for dual ESI18 QFT-ICR MS19 of intact proteins using the
same internal calibrants.
3.3.2 Direct Intact and Product Ion Analysis of Polypeptides by MALDESI-QFT-
ICR-MS
MALDESI-QFT-ICR MS of melittin was performed and multiply-charged ions
were observed. The [M + 4H+]4+ charge-state was isolated and fragmented with the
resultant product-ion spectrum shown in Figure 3.3. The [M + 4H+]4+ charge-state
was chosen so that it could be readily compared with previously recorded nanoESI
data (data not shown); only the y-ion series was observed which is consistent with
19 our previous results. The y13 ion was the predominant species in the product ion
spectrum, due to the propensity of peptides and proteins to cleave to the N-terminal
side of proline residues.20
A rapidly emerging and powerful approach of identifying proteins is intact mass measurement combined with top-down sequence-tag generation.21 Using the
intact mass of the precursor ion, masses of the product ions and the charge-state, a
partial protein sequence can be determined.22 QFT-ICR mass spectrometry is
particularly useful for this type of analysis due to the ability to directly determine the
44 Melittin y17 y16 y15 y13 y12 y11 G I G A V L K V L T T G L P A L I S W I K R K R Q Q
50/50 ACN/Water QFT-ICR MS 0.1% Formic acid Inlet
DHB 83.5 + y13 Melittin
[Melittin + 4H+]4+ Arb. units
3+ y13 Absolute Abundance Absolute y y 11 y12 15 y16 y17 0 500 825 1150 m/z Figure 3.3. Amino acid sequence for melittin with the y11-y13, and y15-y17 fragment ions depicted and the MALDESI QFT-ICR tandem MS fragmentation spectrum of the [M + 4H+]4+ melittin precursor ion. The precursor ions were ionized by MALDESI and accumulated for 2 seconds before mass selection and collision induced dissociation.
charge-state of isotopically resolved clusters,11 and it’s high mass measurement accuracy.23 Future top-down studies of higher molecular weight proteins involving coupling the MALDESI source with QFT-ICR MS will be invaluable as they will allow the identification of proteins produced by direct ionization methods from complex matrices (e.g., tissues).
45 3.3.3 Sensitivity Determination Using the MALDESI Source
In order to begin assessing the limits-of-detection (LODs) for MALDESI-QFT-
ICR MS, a 1 µM solution of SIL angiotensin I (SIL Ang I) was continuously electrosprayed simultaneously with the matrix-assisted laser desorption of naturally occurring angiotensin I (Ang I) prepared in DHB; 2 μL were deposited on a stainless steel MALDI target, resulting in a 4.5 mm diameter sample spot. The resulting
MALDESI-QFT-ICR mass spectrum is shown in Figure 3.4.
Angiotensin I D R VHYIH PLF
QFT-ICR-MS 13 Labeled Labeled C O Inlet angiotensin I * angiotensin I * V MW 1302 13C 13C 13 13C C Angiotensin I + 15 DHB N [Angiotensin I* + 2H+]2+ 101.9 [Angiotensin I + 2H+]2+ Doped nanoESI MALDESI 13 fmoles
Arb. units
Abundance Absolute
0 648 m/z 653 Figure 3.4. Amino acid sequence of angiotensin I and stable isotope labeled angiotensin I (13C515N substituted valine). Representative QFT-ICR mass spectrum of simultaneous MALDESI of natural and nanoESI doped labeled (*) angiotensin I. The average absolute abundance ratio was found and together with the signal to noise ratio was used to calculate the amount of angiotensin I detected.
46 To estimate the LOD of the naturally occurring Ang I by MALDESI-QFT-ICR
MS, we calculated the femtomoles equivalent of SIL Ang I generated in the ESI process by assuming that a 1 µM solution of SIL Ang I electrosprayed at 400 nL/min will generate 13 femtomoles of SIL Ang I ions over the 2 second accumulation window of the instrument. This, of course, is a conservative estimate given that the large majority of electrosprayed ions (>99%) never make it into the entrance orifice of the mass spectrometer for analysis.
The control experiment was conducted between scans without the laser ablation event and the abundance of the electrosprayed SIL Ang I was found to remain constant, demonstrating the lack of concentration effects on the Ang I spectra. The monoisotopic peaks of the [M + 2H+]2+ charge states were used to determine the absolute abundance for Ang I and SIL Ang I. The average absolute abundance ratio of Ang I to SIL Ang I was determined to be approximately 1:1, and used to estimate the amount of Ang I (13 femtomoles). The LOD is inferred to be significantly lower than the 13 femtomoles detected, based on the absolute abundance observed and the signal-to-noise (S/N) in the mass spectrum. The average S/N ratio was calculated (S/N = 63/1) then using the accepted limits of detection (S/N =3/1) the limit of detection was estimated to be 630 attomoles.
47 3.4 Conclusions
Internal calibration is a valuable tool and is readily implemented using
MALDESI by doping the mass calibrant into the ESI solution. The internal calibrant
and analyte are measured simultaneously within the ICR cell which circumvents ion population differences (space charge) commonly found using external calibration and results in mass measurement accuracies of approximately 1 part-per-million.
Intact mass and collision induced fragmentation analyses have been demonstrated using multiply-charged ions generated by MALDESI coupled to a QFT-ICR mass spectrometer. This demonstrates the potential for intact and top-down
characterization of proteins directly from biological matrices (tissues) which should prove to be invaluable. Doping stable isotope labeled analogues (isotopologues)
into the ESI solution provides a strategy by which experimental conditions can be
quantitatively assessed for the direct analysis of peptides and proteins generated
using MALDESI. We are currently investigating matrix crystal morphology using
scanning electron microscopy and determining the exact amount of material ablated
using a quartz crystal microbalance. Additionally, we are investigating methods to
increase the capture efficiency of MALDESI by coupling a modified air amplifier24-27 to decrease our detection limits for direct analysis of peptides and proteins as well as other biological molecules.
48 3.5 References
1. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., Electrospray Ionization for Mass-Spectrometry of Large Biomolecules. Science 1989, 246, 64-71.
2. Karas, M.; Hillenkamp, F., Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10000 Daltons. Analytical Chemistry 1988, 60, 2299-2301.
3. Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T., Protein and Polymer Analysis up to m/z 100 000 by Laser Ionization Time-of-Flight Mass Spectrometry. Rapid Communications in Mass Spectrometry 1988, 2, 151- 153.
4. Lee, C. C.; Chang, D. Y.; Jeng, J.; Shiea, J., Generating multiply charged protein ions via two-step electrospray ionization mass spectrometry. Journal of Mass Spectrometry 2002, 37, 115-117.
5. Coon, J. J.; McHale, K. J.; Harrison, W. W., Atmospheric pressure laser desorption/chemical ionization mass spectrometry: a new ionization method based on existing themes. Rapid Communications in Mass Spectrometry 2002, 16, 681-685.
6. Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G., Mass spectrometry sampling under ambient conditions with desorption electrospray ionization. Science 2004, 306, 471-473.
7. Cody, R. B.; Laramee, J. A.; Durst, H. D., Versatile new ion source for the analysis of materials in open air under ambient conditions. Analytical Chemistry 2005, 77, 2297-2302.
8. McEwen, C. N.; McKay, R. G.; Larsen, B. S., Analysis of solids, liquids, and biological tissues using solids probe introduction at atmospheric pressure on commercial LC/MS instruments. Analytical Chemistry 2005, 77, 7826-7831.
9. Huang, M. Z.; Hsu, H. J.; Lee, J. Y.; Jeng, J.; Shiea, J., Direct Protein Detection from Biological Media through Electrospray-Assisted Laser Desorption Ionization/Mass Spectrometry. Journal of Proteome Research 2006, 5, 1107-1116.
10. Shiea, J.; Huang, M. Z.; Hsu, H. J.; Lee, C. Y.; Yuan, C. H.; Beech, I.; Sunner, J., Electrospray-assisted laser desorption/ionization mass spectrometry for
49 direct ambient analysis of solids. Rapid Communications in Mass Spectrometry 2005, 19, 3701-3704.
11. Henry, K. D.; Williams, E. R.; Wang, B. H.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. F., Fourier-Transform Mass-Spectrometry of Large Molecules by Electrospray Ionization. Proceedings of the National Academy of Sciences of the United States of America 1989, 86, 9075-9078.
12. Marshall, A. G.; Guan, S. H., Advantages of high magnetic field for Fourier transform ion cyclotron resonance mass spectrometry. Rapid Communications in Mass Spectrometry 1996, 10, 1819-1823.
13. Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C., Generation and Detection of Multiply-Charged Peptides and Proteins by Matrix-Assisted Laser Desorption Electrospray Ionization (MALDESI) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Journal of the American Society for Mass Spectrometry 2006, 1712-1716.
14. Frahm, J. L.; Howard, B. E.; Heber, S.; Muddiman, D. C., Accessible proteomics space and its implications for peak capacity for zero-, one- and two-dimensional separations coupled with FT-ICR and TOF mass spectrometry. Journal of Mass Spectrometry 2006, 41, 281-288.
15. Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D. H.; Marshall, A. G., External accumulation of ions for enhanced electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Journal of the American Society for Mass Spectrometry 1997, 8, 970-976.
16. Hannis, J. C.; Muddiman, D. C., A dual electrospray ionization source combined with hexapole accumulation to achieve high mass accuracy of biopolymers in fourier transform ion cyclotron resonance mass spectrometry. Journal of the American Society for Mass Spectrometry 2000, 11, 876-883.
17. Muddiman, D. C.; Oberg, A. L., Statistical evaluation of internal and external mass calibration laws utilized in Fourier transform ion cyclotron resonance mass spectrometry. Analytical Chemistry 2005, 77, 2406-2414.
18. Nepomuceno, A. I.; Muddiman, D. C.; Bergen, H. R.; Craighead, J. R.; Burke, M. J.; Caskey, P. E.; Allan, J. A., Dual electrospray ionization source for confident generation of accurate mass tags using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry. Analytical Chemistry 2003, 75, 3411-3418.
50 19. Williams, K. D., jr.; Hawkridge, A. M.; Muddiman, D. C., Sub Parts-Per-Million Mass Measurement Accuracy of Intact Proteins and Product Ions Achieved using a Dual Electrospray Ionization Quadrupole Fourier Transform Ion Cyclotron Resonance Mass Spectrometer. Journal of the American Society for Mass Spectrometry 2006, Submitted.
20. Loo, J. A.; Edmonds, C. G.; Smith, R. D., Primary Sequence Information from Intact Proteins by Electrospray Ionization Tandem Mass-Spectrometry. Science 1990, 248, 201-204.
21. Kelleher, N. L.; Lin, H. Y.; Valaskovic, G. A.; Aaserud, D. J.; Fridriksson, E. K.; McLafferty, F. W., Top down versus bottom up protein characterization by tandem high-resolution mass spectrometry. Journal of the American Chemical Society 1999, 121, 806-812.
22. Kelleher, N. L., Top-down proteomics. Analytical Chemistry 2004, 76, 196A- 203A.
23. Reid, G. E.; McLuckey, S. A., 'Top down' protein characterization via tandem mass spectrometry. Journal of Mass Spectrometry 2002, 37, 663-675.
24. Zhou, L.; Yue, B. F.; Dearden, D. V.; Lee, E. D.; Rockwood, A. L.; Lee, M. L., Incorporation of a venturi device in electrospray ionization. Analytical Chemistry 2003, 75, 5978-5983.
25. Hawkridge, A. M.; Zhou, L.; Lee, M. L.; Muddiman, D. C., Analytical performance of a venturi device integrated into an electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer for analysis of nucleic acids. Analytical Chemistry 2004, 76, 4118-4122.
26. Yang, P. X.; Cooks, R. G.; Ouyang, Z.; Hawkridge, A. M.; Muddiman, D. C., Gentle protein ionization assisted by high-velocity gas flow. Analytical Chemistry 2005, 77, 6174-6183.
27. Wu, S.; Zhang, K.; Kaiser, N. K.; Bruce, J. E., Incorporation of a flared inlet capillary tube on a Fourier transform ion cyclotron resonance mass spectrometer. Journal of the American Society for Mass Spectrometry 2006, 17, 772-779.
51 Chapter 4
Construction of a Versatile High Precision Ambient Ionization Source for Direct Analysis and Imaging
4.1 Introduction
The introduction and development of hybrid ambient ionization sources such
as laser desorption atmospheric pressure ionization (LDAPI)1, 2, fused droplet
electrospray ionization/extraction electrospray (FD-ESI, EESI)3, 4, direct analysis in
real time (DART)5, desorption electrospray ionization (DESI)6, electrospray assisted
laser desorption ionization (ELDI)7-11, matrix-assisted laser desorption electrospray
ionization (MALDESI)12, 13 and infrared laser desorption electrospray ionization 14, 15 have advanced the capabilities of mass spectrometry. MALDESI, for example, is a pulsed ionization source that holds promise in areas ranging from top-down proteomics, tissue imaging, and ionization mechanism elucidation. The pulsed nature of MALDESI combined with its ability to generate multiply-charged ions are characteristics that are particularly well suited for Fourier transform ion cyclotron resonance (FT-ICR) and Orbitrap (LTQ-Orbi) mass spectrometry due to the inverse relationship of frequency to m/z and √(m/z), respectively. The consequence of this relationship is high resolving power, < 3 ppm mass measurement accuracy, and amenability to a multitude of tandem MS/MS techniques for bottom-up and top-down proteomics (e.g., CID, ETD, ECD, SORI, and IRMPD).16-18 Many of these MS/MS
techniques in FT-ICR and Orbitrap instruments would benefit from a pulsed
52 ionization source where intact proteins and polypeptides with complex post- translational modifications could be carefully interrogated rather than the typical
“continuous” ionization encountered during a LC-MS/MS analysis where peak widths
(i.e. analysis times) are on the order of 10-30 seconds.
The advancement and acceptance of MALDESI and related hybrid ionization techniques are critically dependent on the widespread dissemination of detailed source designs such that results can be reproduced and improved in a variety of laboratories. Furthermore, there are certainly unanticipated potential applications that could benefit from novel or improved source designs. Herein we provide a detailed description and characterization of the third version of the MALDESI source utilized in this laboratory. The further improvement to this design both within our lab as well as other laboratories should enable MALDESI and related hybrid ambient ionization sources to mature to a level of analytical robustness now widely enjoyed for ESI and MALDI.
4.2 Experimental
4.2.1 Materials
Bradykinin, angiotensin I, melittin, glucagon, bovine ubiquitin, lysozyme, myoglobin and 2,5-dihydroxybenzoic acid were purchased from Sigma-Aldrich (St.
Louis, MO, USA) and used without further purification. HPLC-grade acetonitrile and high purity water were purchased from Burdick and Jackson (Muskegon, MI, USA).
53 The electrospray solution was prepared by mixing acetonitrile and water 1:1 (v:v).
The organic matrix solution was prepared by dissolving 150 mg DHB into 1 mL of
the electrospray solution. All samples were prepared from 200 μM stock solutions
and mixed 1:1 (v:v) with the matrix solution. Sample spots of 0.8 μL of the analyte
matrix solution were deposited for each sample, equivalent to ~80 picomoles analyte
per spot.
4.2.2 Profilometery Measurements
A Tencor Alpha Step profilometer (5 nm resolution) was used to measure the
diameter of the laser ablation crater in a gold coated QCM electrode. The distance
was measured across the width of the laser ablation craters with 3 repeats at a scan speed of 50 µm/second. The QCM electrode (International Crystal Manufacturing,
Oklahoma City, OK, USA) consisted of 10 MHz A/T cut quartz crystal with a 1000 angstrom gold layer coated on a chromium base layer.
54 4.2.3 MALDESI-LTQ-FT Mass Spectrometry
MALDESI mass spectra were obtained using a hybrid LTQ-FT Ultra mass spectrometer (Thermo Electron Inc., San Jose, CA, USA and Bremen, Germany) equipped with an actively-shielded 7 T superconducting magnet (Oxford
Instruments, Concord, MA, USA). The MALDESI source (Figure 4.1) was placed in
LTQ-FT mass spectrometer inlet
High magnification CCD camera (Parts#32 - 34)
Syringe pump (Part#28)
Monitor (Part#31)
349 nm UV laser (Part#1)
Vibrationally dampened isolated breadboard (Part #19)
Laptop computer for laser and XYZ control (Part#30)
XYZ motorized stage control (Part#6)
High voltage power supply (Part#27)
Passive isolation support frame (Part#20)
Figure 4.1: Photograph of the new MALDESI ionization source coupled to a LTQ-FT mass spectrometer with major parts indicated and part numbers that correspond to those listed in Table 5.1.
front of the LTQ-FT fitted with a modified extended ion transfer capillary (Part#54).
Solvent was electrosprayed at 2.8 kV through a 75 μm i.d. fused silica capillary
55 (Part#40) with a 30 μm fused silica tapered PicoTip (Part#44) using a Harvard PHD-
2000 syringe pump (Part#28) at a flow rate of 400 nL/minute. The analyte was laser
desorbed from each sample spot actively-dried19 onto a stainless steel sample target
(Part#36) located directly below and between the ESI emitter and the ion transfer
capillary. The stainless steel sample target was biased at 300 volts. Each mass
spectrum was single-acquisition with resolving power at 400 m/z set to 200,000fwhm and the AGC is set to 1 x 106.
4.3 Source Design
4.3.1 MALDESI Source Design and Construction
A new version of the MALDESI source was constructed based in part on the previous source design.12, 13 A photo of the new source is shown in Figure 4.1 with
major parts indicated. A complete parts list, grouped by manufacturer, is included in
Table 4.1, with specifications for custom fabricated parts described in Table 4.2.
56 Table 4.1: MALDESI Parts List
Label Description Distributor Part # Quantity 1 Explorer Q-Switched DPSS Laser Newport (Irvine, CA) EXPL-349-120-1KE 1 2 High performance low profile linear stage Newport (Irvine, CA) 436 3 3 Motorized actuator (X and Y axes) Newport (Irvine, CA) LTA-HS 2 4 Vernier Micrometer (Z axis) Newport (Irvine, CA) SM-50 1 5 Angle bracket (90 deg) Newport (Irvine, CA) 360-90 1 6 2 Axis motion controller/driver Newport (Irvine, CA) ESP300-11N1N1 1 7 19 in. rack mount brackets (ESP300) Newport (Irvine, CA) ESP300-R 1 8 Slotted base Newport (Irvine, CA) B-05A 3 9 2 in. post holder Newport (Irvine, CA) VPH-2 3 10 2 in. ss post Newport (Irvine, CA) SP-2 1 11 4 in. ss post Newport (Irvine, CA) SP-4 1 12 6 in. ss post Newport (Irvine, CA) SP-6 2 13 12 in. ss post Newport (Irvine, CA) SP-12 2 14 Right angle post connector Newport (Irvine, CA) CA-1 1 15 Adjustable angle post connector Newport (Irvine, CA) CA-2 1 16 1 in. lens mount Newport (Irvine, CA) LH-1 1 17 UV fused silica plano convex lens AR10 Newport (Irvine, CA) SPX017 + AR.10 1 18 UV enhanced aluminum mirror Newport (Irvine, CA) 10D20AL.2 2 19 Performance plus breadboard Thorlabs (Newton, NJ) PBI11111 1 20 Passive support frame (27.5"H x 36"L x 30"W) Thorlabs (Newton, NJ) PFP51505 1 21 Periscope assembly Thorlabs (Newton, NJ) RS99 1 22 4 in. stainless steel post Thorlabs (Newton, NJ) RS4 1 23 4 in. mounting post Thorlabs (Newton, NJ) P4 4 24 Swivel casters (3 in.) Grainger 1G196 4 25 Caster brake kit Grainger 4X698 4 26 Power strip (6 plug) Grainger 6X953 2 27 DC power supply Analytica of Branford 103510 1 28 PHD 2000 Syringe pump Harvard Apparatus 70-2000 1 29 Fiber-Lite Light source Dolan Jenner MI-150 1 30 Dell laptop computer (Pentium M, 1.4 GHz) Dell N/A 1 31 Dell 19 in. monitor Dell N/A 1 32 CCD Camera Hitachi KP-M1AN 1 33 Leica optical amplifier Vashaw Scientific 312996 1 34 Leica Monozoom 7 N/A N/A 1 35 Happauge Win-TV USB-2 Circuit City N/A 1 36 SS 192 well MALDI plate Applied Biosystems 4333375 1 37 RS232 to USB converter Tiger Direct N/A 1
57 Table 4.1 Continued 38 High Voltage wire (5kV, 2 pcs. 12 in., 24 in.) N/A N/A 1 39 LTQ high voltage plug Connectronics Corp. 10334-02 1 40 24 in. fused silica capillary (75 um i.d.) Polymicro Technologies 2000019 1 41 Stainless steel union Valco Instrument Co. ZUIXC 1 42 Syringe adapter Valco Instrument Co. VISF-2 1 43 PEEK tubing sleeve orange 0.062x0.016 Upchurch Scientific F-230 2 44 Silica tip (30um i.d. tapered) New Objective Inc. FS360-75-30-N-20 1 45 Hamilton gas tight syringe 100 uL Fisher Scientific 14-813-138 1 46 Breadboard Fabricated in house N/A 1 47 Camera support bracket Fabricated in house N/A 1 48 Teflon ESI holder Fabricated in house N/A 1 49 Sample target high voltage clip Fabricated in house N/A 1 50 19 in. rack support bracket Fabricated in house N/A 2 51 Sample target right angle bracket Fabricated in house N/A 1 52 Sample plate Teflon insulator Fabricated in house N/A 1 53 Computer shelf Fabricated in house N/A 1 54 Extended capillary Fabricated in house N/A 1 55 Laser spacing block Fabricated in house N/A 1
Table 4.2: Description of parts fabricated in-house 16 in. x 16 in. x 3/8 in. thickness aluminum (thickness may be reduced to 1/4 in.), drilled and tapped 1/4 x 20 (1" on 46 center)
47 8 in. x 1/2 in. thickness aluminum, two piece clamp for Monozoom lens support
2 in. x 1/2 in. x 1/4 in. thickness Teflon; cut-out to receive a stainless steel union, threaded tube inserted across width to 48 attach aluminum retainer and electrical contact for ESI voltage
49 1¼ in. x 1/8 in. x 1/32 in. thickness stainless steel with custom bend for electrical contact with sample target
16 in. x 2½ in. (1½ in. along component attachment region) x 1/4 in. thickness aluminum, slots at each end for 50 attachment to support frame (Part#20) 3 in. length x 2 ½ in. height, 1/4 in. thickness aluminum, (2) vertical slots cut in short side for attachment to XYZ stage, (2) 51 clear holes (#8) in long side for Teflon target insulator attachment 2½ in. x 2½ in. x 1/4 in. thickness Teflon with (2) small stainless steel posts to accommodate the sample target and (2) 52 holes drilled and tapped 8/32 in. from bottom to attach to sample target bracket (Part#51)
53 1/4 in. thickness aluminum, cut to fit across upper cross members of support frame (Part#20)
54 2 in. extended stainless steel capillary with lug to fit LTQ atmospheric pressure interface
7½ in. x 3¾ in. x 1½ in. aluminum block including ½ in. slots on each end for ¼ x 20 screw attachment to the breadboard 55 (Part#19)
58 A schematic of the ionization source labeled by part number is included in
Appendix A (Figure 4S1). The manual linear XYZ sample positioning stages were
replaced with computer controlled motorized sample positioning stages (Parts#2-5)
and control unit (Part#6) for precise sample positioning during analysis. The high
speed long travel translational stages (Parts#2, 3) allow for interrogation of samples
across the full length and width of a standard MALDI target (Part#36). Custom
translation programming was accomplished using the supplied software (Newport,
ESP); enabling one touch program execution for whole sample spot analysis (a
diagram of the ablation path is shown in Appendix A (Figure 4S2)) as well as
allowing the user to operate the source remotely. The position control can be
actuated linearly without program execution for spot to spot analysis, as typically
performed in MALDI. The high precision (0.035 µm resolution, 100 nm increments)
of the positioning system enables accurate repeatable (+ 600 nm, bi-directional)
positioning amenable to applications requiring a high degree of positioning control
(e.g., tissue imaging).
The manually actuated pulsed nitrogen laser (337 nm) was replaced by the
Explorer laser system (Part#1), a Q-switched diode pumped solid-state ultraviolet
laser (349 nm) with first pulse suppression, precise internally measured laser power
(0-120 μJ) and repetition rate control (0-5000 Hz) using the supplied software
(Spectra-Physics, L-Win). The laser power, repetition rate and translation stage velocity control (0-5 mm/s) are invaluable for applications to various samples,
59 surfaces and desorption conditions; preventing damage to solid substrate and
depletion of analyte during analysis. The high resolution CCD camera (Parts#32-34)
was connected using the WinTV PCI tuner (Part#35) to the laptop computer and
monitor (Parts#30, 31) enabling real time on screen visualization and recording of the sample spot and tracking of the laser spot on target during laser ablation, which is particularly useful for analysis of small sample spots as well as tissue sections for imaging.
The XYZ stage (Parts#2-5, 38, 49, 51, 52), second stage laser beam positioning mirror (Part#18, mounted in the upper portion of the periscope assembly
Parts#21, 11, 8), focusing lens assembly (Parts#8, 9, 11, 13, 14, 16, 17), ESI emitter assembly (Parts#8, 9, 12, 12, 15, 40-44, 48) and CCD camera assembly (Parts#8, 9,
10, 13, 32-34, 47) were all mounted onto the custom fabricated breadboard
(Part#46).
A schematic of the desorption and ionization region with a description of each sub-assembly are shown in Figures 4.2 and 4.3. A clear hole drilled in the
breadboard (Part#46) allows the passage of the laser beam from the first stage
directional mirror (Parts#18, 21, 22) mounted on the main breadboard work surface
(Part#19) to the second stage directional mirror (Parts#8, 11, 18, 21) mounted on
the custom breadboard (Part#46). The breadboard (Part#46) with all mounted equipment was then mounted onto the vibrationally isolated main breadboard
(Part#19) using 4 inch offset posts (Part#23). The laser (Part#1) mounted to a
60 custom fabricated spacing block (Part#55), first stage laser beam positioning mirror
(Part#18 mounted in lower portion of the periscope assembly, Part#21, attached to a
UV Laser CCD Camera (Parts# 32 - 34) 5 mm Directional mirror nd (2 Stage, Parts# 8, 11, 18, 21) 45o 3 mm
50 V 300 V 2.5 kV LTQ-FT MS inlet
Target platform ESI emitter (Parts# 38, 49, 51, 52) Laser (Parts# 1, 55) Directional mirror (1st Stage, Parts# 18, 21, 22) Breadboard (Part#19) Focusing lens not shown
Figure 4.2: Side view schematic of the ionization region of the MALDESI source with the numbers corresponding to the parts listed in Table 5.1.
4 inch post, Part#22), syringe pump (Part#28), light source (Part#29) and monitor
(Part#31) were mounted on the main breadboard (Part#19). The field of the main breadboard (Part#19) and custom breadboard (Part#46) were fabricated with tapped
holes (1/4 x 20) on one inch center; a supply of appropriate fasteners is required to
attach various components to the breadboards.
The high voltage power supply (Part#27) and motion controller (Part#6) were
mounted to the support frame (Part#20) using custom fabricated rack mounting
brackets (part#50). The high voltage power supply (Part#27) was attached to the
61 sample target high voltage clip (Part#49) using 5 kV insulated wire (Part#38) for
contact with the stainless steel sample target (Part#36) mounted on the Teflon
insulator (Part#52). The keyboard, mouse and laptop computer (Part#30) were
placed onto a custom fabricated shelf (part#53) situated on the upper cross beams
of the support frame (Part#20). Power management was provided using two 6 plug
power strips (Part#26) installed on the inside of the vertical supports on the rear of
the support frame (Part#20). Prior to assembly, casters (Parts#24, 25) were installed
to replace the existing adjustable feet on the support frame for mobility. The versatility of this ionization source platform allows for interchangeability of the source
between instruments as well as installation of new components onto the existing
platform.
62 Syringe Pump To Power Supply (Part#27) (Part#28) HV wire (Part#38) Breadboard (Part#46)
LTQ ESI HV plug ESI assembly: (Part#39) Y X SilicaTip (Part#44), Peek tubing (Part#43), ss union (Part#41), Peek tubing (Part#43), fused silica capillary (Part#40), syringe adaptor (Part#42) Z Teflon holder (Part#48), 6 in. ss post (Part#12), adjustable angle post LTQ-FT connector (Part#15), 6 in. ss post MS inlet (Part#12), post holder (Part#9), base (Part#8)
Laser (Part#1), Extended Capillary spacing block (Part#54) (Part#55)
2nd stage directional mirror assembly: Focusing lens assembly: Mirror (Part#18), top portion periscope assembly Focusing lens (Part#17), lens mount (Part#16), (Part#21, small adj. knob removed for mirror 4 in. ss post (Part#11), right angle post positioning), 6 in. ss post (Part#12), 4 in. ss post connector (Part#14), 12 in. ss post (Part#13), (Part#11), post holder (Part#9), base (Part#21) post holder (Part#9), base (Part#8) CCD camera assembly (not shown): CCD camera (Part#32), optical amplifier (Part#33), lens stack (Part#34), custom bracket (Part#47), 12 in. ss post (Part#13), 2 in. ss post (Part#10), post holder (Part#9), base (Part#8)
SilicaTip (Part#44) Stainless steel union (Part#41) Fused silica capillary (Part#40)
Teflon ESI holder (Part#48) 12 in. ss post (Part#13) 6 in. ss post (Part#12)
Figure 4.3: Top view schematic of the ionization region with a listing of the parts for each sub-assembly.
63 4.3.2 Desorption and Ionization Source Versatility
The high stability ionization source platform may be implemented using
various desorption and ionization techniques for application specific analysis.
Existing ionization sources may be installed onto the platform due to the inherent
versatility of the breadboard for roll-up accessibility. Analyte desorption may be
induced by one of a number of regimes including laser desorption as in MALDESI,
laser induced acoustic desorption (LIAD),20 heated nitrogen gas as in ASAP21 and
impact of high velocity charged droplets as in DESI6 followed by a number of post- desorption ionization methods including atmospheric pressure photo ionization
(APPI)22, atmospheric pressure chemical ionization (APCI)23 and electrospray
ionization (ESI)24. In addition to the hybrid ionization sources listed above, this platform is amenable to basic electrospray ionization24 and atmospheric pressure
matrix-assisted laser desorption ionization.25
64 4.4 Results and Discussion
4.4.1 Explorer Laser Beam Characterization
The laser beam spot size was measured at a laser power of 112 µJ
(measured internally) using a gold coated A 1.2 μm 6400Å 60 μm quartz crystal microbalance electrode as the
2200Å Height
-2000Å laser target. A photo of the QCM electrode 150 187 224 Scan length (μm) following laser ablation is shown in Figure B 4.4B, with an expanded view of a single
ablation crater shown in Figure 4.4C. In these C
80 μm experiments, the thin gold top layer (1000
60 μm angstroms) was removed exposing the
Figure 4.4: A) Profilometer chromium base layer with some re-deposition scan plot across the width (60 µm) of the laser ablation crater along the perimeter of the ablated crater. The in the gold QCM electrode. B) Photograph of the QCM elliptical laser beam spot size (60 µm × 80 µm) electrode after laser desorption. C) Expanded view of an was determined by measuring the actual elliptical ablation crater, the diameters (60 µm x 80 µm) were diameter of the ablation craters using a Tencor measured for multiple ablation craters across the QCM Alpha Step profilometer. A representative scan electrode. plot is shown in Figure 4.4A.
65 4.4.2 MALDESI-LTQ-FT-ICR of Peptides and Proteins
MALDESI-FT-ICR mass spectra of peptides and proteins (1 – 8.6 kDa)
including bradykinin, angiotensin I, melittin, glucagon and ubiquitin each mixed with
organic matrix have been shown previously.12, 13 Comparable data was obtained
using the new source; representative MALDESI-FT-ICR mass spectra of bradykinin
and melittin both mixed with DHB included in Appendix A (Figure 4S3). Lysozyme
and myoglobin (not shown previously) were each mixed 1:1 (v:v) with DHB, 0.8 μL
was deposited onto the sample target and actively dried.19 The ESI solution flow-rate
was set to 400 nL/minute and stable electrospray was obtained. The motion
controller was pre-programmed to raster the sample surface under the laser beam
as illustrated in the Appendix A (Figure 4S2). The laser was actuated using
computer control at a laser power of 50 μJ (measured internally) and repetition rate
of 10 Hz and the motion program initiated. Multiply-charged ions were generated and detected for each peptide and protein; demonstrating a molecular weight range
from 1 – 17 kDa using this source. Representative MALDESI-FT-ICR mass spectra
of myoglobin and lysozyme from this experiment are shown in Figure 4.5, with an
observed resolving power of ~ 40,000.
The amount of material ablated during analysis was calculated using the
amount of material spotted (~80 picomoles), collection time (200 ms), laser repetition
rate (10 Hz, 2 shots per collection), area of the laser spot (3846 μm2) and the area of
the dried spot (A = 3.14 mm2); 196 femtomoles were removed per spectrum,
66 assuming [M + 15H+]15+ A 45181 +]16+ [M + 16H Myoglobin uniform
[M + 14H+]14+ distribution of
analyte and [M + 17H+]17+
[M + 13H+]13+ complete Absolute Abundance Absolute [M + 18H+]18+ removal at each [M + 19H+]19+ [M + 20H+]20+ [M + 12H+]12+ laser shot. The [M + 11H+]11+ 0 development 800 1200 1600 m/z [M + 11H+]11+ B 1169 and
+ 10+ [M + 10H ] implementation Lysozyme of molecular [M + 9H+]9+ transport [M + 12H+]12+ Absolute Abundance Absolute devices such as
+ 8+ [M + 8H ] the air
26, 27 0 amplifier, air 1000 1500 2000 m/z ejector (AE)28 Figure 4.5: MALDESI-FT-ICR mass spectra of 0.8 μL actively dried spot of 200 μM A) myoglobin (16.9 kDa) and B) lysozyme C (14.3 kDa) mixed 1:1 and RASTIR29 (v:v) with 150 mg/mL DHB.
which enable the efficient transport of ions into the mass spectrometer have been investigated and may prove important to increasing sensitivity.
67 4.4.3 Liquid Drop Sample Analysis
Liquid drop analysis of 0.8 μL droplets of 200 μM ubiquitin and myoglobin mixed 1:1 (v:v) with DHB (150 mg/mL) and deposited onto the sample target, biased at 300 V, for immediate analysis is demonstrated using MALDESI with ESI post- ionization. Liquid drop laser desorption analysis with ESI post-ionization has been
demonstrated previously.11, 30 The ESI emitter was biased at 2.8 kV to electrospray
50 % acetonitrile in water at a flow rate of 400 nL/minute. The liquid sample was
continuously irradiated using the Explorer laser without moving the sample target,
yielding relatively constant ion abundance over the lifetime of the droplet
(approximately 30 seconds). liq-MALDESI-FT-ICR mass spectra of myoglobin and
ubiquitin with ESI post-ionization are shown in Appendix A (Figure 4S4), A and B,
respectively.
4.5 Conclusions
The MALDESI ionization source described herein couples high stability with
precision motion and laser control. This design enables high mass resolving power analysis of biological molecules including intact and top-down characterization in addition to facilitating potential imaging applications.
The mobile design provides a stable analytical platform which may be used to implement a number of desorption regimes including laser desorption (e.g.,
MALDESI), high velocity solvent droplets (e.g., DESI), a stream of heated nitrogen
68 gas (e.g., ASAP) and post desorption ionization using electrospray ionization (ESI), chemical (e.g., APCI) and photon ionization (e.g., APPI) for analysis of various types and classes of molecules on multiple MS platforms. The modular configuration of the
MALDESI ionization source can facilitate the substitution of parts as necessary for specific applications or budgeting constraints. Furthermore, an IR laser could be mounted to the laser table breadboard for infrared laser MALDESI applications (IR-
LDESI).14, 15
69 4.6 References
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20. Golovlev, V. V.; Allman, S. L.; Garrett, W. R.; Taranenko, N. I.; Chen, C. H., Laser-induced acoustic desorption. International Journal of Mass Spectrometry 1997, 169, 69-78.
21. McEwen, C. N.; McKay, R. G.; Larsen, B. S., Analysis of solids, liquids, and biological tissues using solids probe introduction at atmospheric pressure on commercial LC/MS instruments. Anal Chem 2005, 77, 7826-7831.
22. Robb, D. B.; Covey, T. R.; Bruins, A. P., Atmospheric pressure photoionization: an ionization method for liquid chromatography-mass spectrometry. Anal Chem 2000, 72, 3653-3659.
23. Shahin, M. M., Mass-Spectrometric Studies of Corona Discharges in Air at Atmospheric Pressures. Journal of Chemical Physics 1966, 45, 2600-&.
24. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., Electrospray Ionization for Mass-Spectrometry of Large Biomolecules. Science 1989, 246, 64-71.
25. Laiko, V. V.; Baldwin, M. A.; Burlingame, A. L., Atmospheric pressure matrix assisted laser desorption/ionization mass spectrometry. Analytical Chemistry 2000, 72, 652-657.
26. Zhou, L.; Yue, B. F.; Dearden, D. V.; Lee, E. D.; Rockwood, A. L.; Lee, M. L., Incorporation of a venturi device in electrospray ionization. Analytical Chemistry 2003, 75, 5978-5983.
27. Dixon, R. B.; Muddiman, D. C.; Hawkridge, A. M.; Fedorov, A. G., Probing the mechanisms of an air amplifier using a LTQ-FT-ICR-MS and fluorescence spectroscopy. J Am Soc Mass Spectrom 2007, 18, 1909-1913.
28. Dixon, R. B.; Bereman, M. S.; Muddiman, D. C.; Hawkridge, A. M., Remote mass spectrometric sampling of electrospray- and desorption electrospray- generated ions using an air ejector. J Am Soc Mass Spectrom 2007, 18, 1844-1847.
29. Dixon, R. B.; Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C., Novel Ambient Aerodynamic Ionization Source for Remote Analyte Sampling and Mass Spectrometric Analysis. Analytical Chemistry 2008, Accepted.
72 30. Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C., Development and characterization of an ionization technique for analysis of biological macromolecules: Liquid matrix-assisted laser desorption electrospray ionization. Analytical Chemistry 2008, 80, 6773-6778.
73 Chapter 5
Development and Characterization of an Ionization Technique for Analysis of Biological Macromolecules: Liquid Matrix-Assisted Laser Desorption Electrospray Ionization
5.1 Introduction
Mass spectrometry has become one of the most powerful techniques for characterization and identification of biological molecules. Since the advent of matrix-assisted laser desorption ionization (MALDI)1, 2 and electrospray ionization
(ESI)3 the overall utility of mass spectrometry has increased significantly, particularly
in the area of biological sciences including proteomics and metabolomics. Recently,
several novel ambient ionization sources have been developed that enable direct
analysis at atmospheric pressure such as laser desorption atmospheric pressure
chemical ionization (LD-APCI),4, 5 desorption electrospray ionization (DESI),6 direct
analysis in real time (DART),7 atmospheric pressure solids analysis probe (ASAP),8 laser induced acoustic desorption (LIAD)9 electrospray assisted laser desorption
ionization (ELDI),10, 11 matrix-assisted laser desorption electrospray ionization
(MALDESI)12, 13 and most recently laser ablation electrospray ionization (LAESI),14 atmospheric pressure matrix-assisted infrared laser desorption/ionization (AP-IR-
MALDI)15 and infrared laser-assisted desorption electrospray ionization (IR-
LADESI).16 The advantages of direct analysis under atmospheric pressure are direct
access to samples during analysis and no additional sample preparation steps
74 required for vacuum sensitive samples (e.g., tissue slices). Laser desorption
ionization sources have the added benefits of minimal sample preparation and high spatial resolution due to the inherently small laser spot sizes which makes them ideal for analysis of biological molecules and tissue imaging applications.17, 18
ELDI, MALDESI and LAESI are based on laser desorption followed by post-
ionization via electrospray ionization. Introduction of an internal calibrant via the ESI
solution has been demonstrated to achieve high mass measurement accuracy of
desorbed ions without interfering with the analyte/matrix co-crystallization of the
sample.13 Recently, liquid sample analysis was demonstrated by ultraviolet laser
desorption from matrix containing liquid samples followed by post-ionization by ESI
using ELDI11 and using infrared laser desorption from liquid samples without ESI
post-ionization using AP-IR-MALDI.15 In a similar method described herein, a
variation of our MALDESI source, liquid matrix-assisted laser desorption
electrospray ionization (liq-MALDESI) utilizes an ultraviolet laser directed onto the
surface of an analyte containing liquid droplet biased at a high potential. In this method, the charged droplet acts as a macroscopic ESI droplet, from which offspring droplets are laser desorbed which then undergo an ESI-like desorption and ionization process generating multiply-charged ions.
Sample preparation using AP-IR-MALDI includes the addition of anhydrous glycerol and heating at 50oC for 20 minutes or placing into a desiccator for 1 hour to
remove excess water prior to analysis. Glycerol is generally is added to increase the
75 viscosity of the solvent/sample, which in this case may impede the desorption of analyte from the droplet possibly leading to the long analysis times and decreased sensitivity (25 times longer analysis time for 6 times the amount of deposited analyte, compared to liq-MALDESI, reported herein) demonstrated by Dreisewerd and coworkers.15 In contrast, sample preparation for liq-MALDESI involves mixing analyte with an ultraviolet absorbing organic matrix, both dissolved in aqueous acetonitrile. No lengthy sample heating or desiccation is required prior to analysis.
Under our laboratory conditions, the surface tension of the liquid droplet impacts both the ion abundance and sensitivity.
In ESI, a high potential is applied to aqueous solution which causes the oxidation of water, generating excess protons in solution. The solution exits through an emitter tip and due to coulombic repulsion charged droplets are expelled from the emitter tip. The surface area of the droplet decreases due to evaporation of solvent,
thereby increasing the charge
Contact angle Liquid-vapor density on the droplet surface. interface +++++++++ The increased charge density +------+- Electrical +++++++++++ double layer results in a decrease in the Applied voltage (3 kV) Sample target surface tension of the charged
Figure 5.1: Diagram of a liquid sample drop solvent droplets which plays a deposited onto a stainless steel sample target biased at 3 kV. The solid-liquid-vapor contact angle key role in the desorption of measurement is demonstrated in addition to the electric double layer at the solid-liquid interface. ions.19 For a static droplet
76 deposited on a surface, as shown in Figure 5.1, the same general principles apply;
an increase in applied potential necessarily increases the number of charges (due to
oxidation of water) on the droplet surface which results in a decrease in the surface
tension of the droplet.
The surface tension for a static droplet on a surface is often determined by
measuring the contact angle made at the three-phase line, the intersection of the
solid, liquid and gas phases. The Gibbs, Johnson and Neumann approach can be
used to relate the surface tension of a liquid drop to the contact angle
measurements under a variety of equilibrium conditions including the addition of
electric charges.20 The decrease in contact angle due to applied potential may be
attributed to the reduction of surface tension.20 Charge separation occurs at the
liquid-vapor interface due to Coulombic repulsions of like charges while an electrical
double layer (EDL) forms at the solid-liquid interface (Figure 5.1) thereby minimizing
the energy of the droplet on the surface.21
Herein, we demonstrate the generation and detection of multiply-charged
peptide and protein ions by liq-MALDESI from liquid sample drops containing analyte mixed with organic matrix deposited onto a stainless steel sample target biased at a high potential. The high potential applied to the sample target was varied to optimize conditions for desorption and ionization, the contact angle was measured across the voltage range to determine the effects of increased applied potential on droplet surface tension and detection using liq-MALDESI.
77 5.2 Experimental
5.2.1 Materials
Bradykinin, melittin, ubiquitin, rubrene, 2,5-dihydroxybenzoic acid (DHB) and dichloromethane were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. High purity water and HPLC grade acetonitrile were purchased from Burdick and Jackson (Muskegon, MI, USA) and used as received.
The contact angle measurements were performed using a rame-hart CA-100-
00 contact angle goniometer equipped with a microscope for silhouette viewing of liquid drops on a surface, two independently rotatable crosshairs and an internal readout protractor for precise contact angle measurements. The 2 μL liquid sample drops were deposited onto a stainless steel MALDI target mounted on a Teflon insulator attached to the goniometer. The sample target potential was applied using a high voltage power supply (Analytica of Branford, Branford, CT, USA).
5.2.2 liq-MALDESI-LTQ and LTQ-FT Mass Spectrometry
A new MALDESI source was constructed based partly on the design described previously,12 with the addition of a high precision computer controlled motion control system (ESP-300, Newport Corporation, Irvine, CA, USA) and a 349 nm UV laser (Explorer, 349-120-1KE, Newport Corporation, Irvine, CA, USA) all mounted to an independent support frame (PFP51505, Thorlabs, Newton, NH,
USA). Two UV enhanced aluminum mirrors and a fused silica convex lens
78 (10D20AL.2 and SPX017+AR.10, Newport Corporation, Irvine, CA, USA) were assembled using a periscope assembly (RS 99, Thorlabs, Newton, NH, USA) onto a vibrationally damped breadboard (PBI11111, Thorlabs, Newton, NH, USA) supported by the passively isolated support frame which also houses the high voltage power supply (Analytica of Branford, Branford, CT, USA), XYZ stage positioning controller and laptop computer.
The high repetition rate (1-5000 Hz) computer controlled Explorer laser was affixed to the breadboard such that the beam could be directed using the directional mirrors and focused onto the sample target directly in front of the mass spectrometer and connected to the onboard laptop computer. The laser repetition rate was set to
50 Hertz at 90 μJ laser power, measured internally. The stainless steel 192 well sample target (4333375, Applied Biosystems, Foster City, CA, USA) was affixed to a
Teflon insulator attached to a XYZ motorized linear stage (436, Newport
Corporation, Irvine, CA, USA) and positioned approximately 5 mm below the MS
inlet capillary. The desorption/ionization region (i.e., sample target) was not enclosed
in any way and remained open to the ambient atmosphere with no temperature
regulation. A high magnification CCD camera was positioned above the sample
plate for live monitoring of the sample during analysis. A full description of the
source construction including a complete parts list is described elsewhere.22
A LTQ linear ion trap and a hybrid LTQ-FT Ultra mass spectrometer, both
from Thermo-Electron (San Jose, CA, USA), were used in these experiments. The
79 LTQ-FT was equipped with an Oxford Instruments 7 Tesla superconducting magnet
and spectra were collected at a resolving power setting of 100,000FWHM at m/z =
400. The ion transfer capillary voltage and temperature were set to 40 V and 200oC, respectively.
5.3 Results and Discussion
5.3.1 liq-MALDESI-LTQ Liquid Sample Analysis
Analyte mixed with matrix was deposited onto the sample target for
immediate (1 – 30 seconds) analysis of the liquid sample. In this experiment, a
reproduction of the work done by Loo’s group,11 the analyte (melittin, 100 μM) mixed
with an organic matrix (DHB, 75 mg/mL) was deposited (0.5 μL) onto the sample target and immediately analyzed by laser ablation of the liquid sample while electrospraying 50% acetonitrile in water. The ESI emitter and stainless steel sample target were biased at 2.6 kilovolts (kV) and 500 volts, respectively. A schematic and representative mass spectrum of melittin are shown in Figure 5.2A. The ion abundances are similar to those obtained by solid-state MALDESI; however, the sample target was not moved during analysis and the shot-to-shot reproducibility was improved yielding long lasting signal (~ 30 seconds/spot).
The laser energy absorbed by the matrix is readily transferred to the surrounding analyte or solvent molecules in the liquid phase thereby enhancing desorption. The improved duration of signal from the liquid drop compared with solid
80 state MALDESI indicates that desorption and/or ionization efficiency are improved for a comparable volume (0.5 μL) and concentration of analyte (100 μM) mixed with
DHB to create a dried sample spot. The control experiment was attempted to exclude desorption of multiply-charged ions from the liquid droplet on the surface by replacing the ESI emitter with a copper electrode biased at the same potential (3000
+ + MS Inlet Capillary + 4+ + + + UV laser 265 [M + 4H ] + + + + + + + + + ++ + + + + + A ++ + + + + + + + + + + + + + + 3+ + + + + [M + 3H ] + + + + + + ESI emitter + + + + ++ 3000 V + + Liquid Sample 40 V + + + + + + + + Na+ Absolute Abundance0 500 volts Solvent 60 MS Inlet Capillary Analyte + + 3+ + + + + UV laser [M + 3H ] + + + + + [M + 4H+]4+ + + DHB B ++ + + ++ + + + + + + + + + + + + + + + + ++ + + + + + + 40 V + + + + + Liquid Sample
+ + + AbundanceAbsolute + + + 0 600 900 1200 3000 volts m/z Figure 5.2: Ionization schemes and representative mass spectra for A) liq-MALDESI from liquid drop containing analyte mixed with matrix while electrospraying 50 % acetonitrile in water and B) liq-MALDESI without ESI post-ionization, the sample target was at biased at 3 kV.
V) by laser desorption of a new liquid drop of the same analyte solution with the sample target biased at 500 V. This experiment yielded a weak multiply-charged analyte signal.
Based on this observation from the control experiment it was evident that the
ESI emitter was not necessary for generation of multiply-charged ions. Therefore we purposefully applied a high-voltage to the sample target in the absence of the ESI
81 emitter or copper electrode. The potential applied to the sample target was increased from 500 V to 3 kV, resulting in an increase in ion abundance. Ion abundances increase up to 2-3 kV, then decreases with additional applied voltage
(up to 5 kV) (vida infra). A schematic of liq-MALDESI and representative mass spectrum of melittin mixed with DHB are shown in Figure 5.2B. Sodium adduction is very prevalent in the liq-MALDESI mass spectra with the ESI emitter removed. The role of the electrosprayed solvent in the ionization process for both solid-state and liq-MALDESI in previous experiments is that it functions as an intermediate, fusing with desorbed neutral analyte molecules (solid-state) or analyte, matrix and sodium containing desorbed droplets (liquid-state) which then undergo an ESI-like desorption and ionization process. Sodium adduction is not encountered in solid- state MALDESI as a result of the exclusion of charged sodium adducted ions from the electrosprayed droplets. In the absence of the ESI plume, multiply-charged ions desorbed from the charged liquid sample drop, including sodium adducted ions are readily sampled by the mass spectrometer. The control experiments were conducted for a liquid droplet on an unbiased target and for dried samples with and without applied voltage (0-5 kV), all without ESI post-ionization, yielding no analyte signal, providing evidence that this is not a pure AP-MALDI process, as suggested by
Dreisewerd.15
82 5.3.2 liq-MALDESI Ion Abundance Versus Sample Target Potential
Liquid drop samples
A Bradykinin 800 of bradykinin, melittin and Melittin Ubiquitin 700 ubiquitin (10 μM mixed with 600
500 DHB) were used to
400 characterize the ion 300 abundance dependence on
Absolute Abundance 200 100 the sample target potential. 0 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Each 0.5 μL sample was Target Potential (kV) deposited onto the biased B 80 Pure Water y = -2.9x + 61.3 0.2 % Formic acid sample target for immediate 70 R2 = 0.980 Melittin + DHB 60 (1 – 30 seconds) analysis by
50 y = -2.4x + 38.6 R2 = 0.968 liq-MALDESI (without ESI 40 post-ionization). The sample 30
20 target bias was varied Contact Angle (Degrees) Angle Contact y = -2.1x + 26.3 10 2 R = 0.964 between 0 and 5000 volts in Decreasing Surface Tension Surface Decreasing 0 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 1000 volt increments and Target Potential (kV) Figure 5.3: Plot of A) absolute abundance versus target mass spectra were collected potential for 10 μM solutions of bradykinin, melittin and ubiquitin mixed with DHB using liq-MALDESI and B) plot utilizing a fresh sample drop of contact angle versus applied target potential for pure water, 0.2% formic acid in water and a solution of melittin at each potential. A plot of (10 μM) mixed with DHB (10 mM). Without ESI post- ionization, sample target bias was varied. the absolute abundance
83 versus the sample plate potential is shown in Figure 5.3A. At a potential of 0 volts,
liq-MALDESI did not yield detectable signal in the mass spectrometer. The ion
abundance for bradykinin and melittin demonstrated a steady increase up to 2 kV
(maximum abundance), whereas for ubiquitin the maximum ion abundance was
observed at 3 kV. The increased potential required for larger molecules may be necessary to increase the surface charge density of the drop, thereby increasing the number of charges available to the analyte facilitating its desorption from the droplet.
One interesting observation is there was no shift in the average charge-state across
the range of applied potentials, only a change in absolute ion abundances.
5.3.3 Contact Angle Versus Sample Target Potential
We hypothesized that with an increase in the applied potential we should
observe a reduction in the surface tension of the drop. A method commonly used to
determine surface tension (surface energy) on a drop is to measure the contact
angle made by the solid, liquid and gas phase interface (three phase contact line) of
the droplet on a surface.
The contact angle was measured for liquid droplets (2 μL) of pure water,
water with 0.2 % formic acid and a sample of melittin (10 μM) mixed with DHB
across the voltage range (0 – 5 kV, 1 kV increments). Each drop was placed onto
the sample target, the contact angle was measured, the potential was increased and
the contact angle was re-measured, this was repeated across the voltage range.
84 These results are shown in Figure 5.3B. The contact angle for each sample decreased for increasing applied potential. The decrease in the contact angle is influenced by the formation of an electric double layer (EDL) at the solid-liquid interface (negative charges align on the liquid side opposite the positive charges on the sample target) and the build-up of positive charges on the liquid-vapor interface.
The EDL results in a reduction in the energy of the solid-liquid interface, allowing more of the droplet surface to interact with the sample target while the positive charge build-up at the liquid-vapor interface corresponds to a decrease in surface tension. The decrease in the surface tension with increasing applied potential supports the theory that charges accumulate on the surface and that laser desorption liberates analyte containing charged droplets which then undergo an ESI- like desorption and ionization process. This data also supports the oxidation of water at the target/water interface.
5.3.4 liq-MALDESI-LTQ-FT-ICR Analysis of Peptides and Proteins
Although sodium adduction is evident in the initial liq-MALDESI mass spectra at high analyte + matrix concentrations, reduced concentrations of analyte mixed with DHB show significantly reduced or no sodium adduction. A 0.5 μL drop of bradykinin, melittin or ubiquitin mixed with DHB was deposited onto the stainless steel sample target biased at 3 kV for immediate (1 – 30 seconds) analysis. The concentration of DHB was varied for each analyte and the optimal ratio was found to
85 be 1:1000 for bradykinin and melittin and 1:2500 for ubiquitin (data not shown). The
analyte-to-matrix ratio range (1:1000 to 1:2500) was acceptable to desorb and ionize
any of the standard peptides and proteins used in these experiments with only
marginal gain/loss in ion abundances across the range for each analyte. Several
spectra were obtained for both bradykinin and melittin at high analyte concentrations
(100 μM) without addition of matrix; however, the ion abundance was low and the
signal was short lived (2-3 spectra/sample drop), relative to samples with matrix. No
signal was obtained from ubiquitin without matrix. It is important to note, as
mentioned previously, no analyte signal was observed after the liquid has
evaporated.
The amount of analyte deposited for analysis was reduced by diluting the
original solution, thereby preserving the optimal analyte-to-matrix ratio. The amount
of analyte deposited onto the surface in a 0.5 μL droplet of a 5 μM solution was 2.5 picomoles (bradykinin and melittin) and 25 picomoles deposited for 0.5 μL of a 50
μM solution (ubiquitin). Representative liq-MALDESI-FT-ICR mass spectra of
bradykinin (5 μM), melittin (5 μM) and ubiquitin (50 μM) are shown in Figure 5.4 A,
B and C. Bradykinin and melittin FT-ICR mass spectra were readily obtained from 5
μM solutions with no sodium adduction as shown in Figure 5.4 A and B; however,
86 for ubiquitin, a 50 μM solution was necessary to achieve appreciable ion abundance, as a result of the increased concentration, the concentration of matrix in the sample
was also A 552 [M + 2H+]2+ increased [M+1H+]1+ (preserving the
analyte-to-matrix Absolute Abundance ratio), contributing 0
B [M + 4H+]4+ to the observed 3907 [M + 3H+]3+ matrix and sodium
adduction. [M + 5H+]5+ In a Absolute Abundance Absolute
0 separate
[M + 10H+]10+ C experiment
2206 [M + 11H+]11+ samples were [M + 2 DHB + Na+ + (n-1)H+]n+ [M + 9H+]9+ * prepared by
+ 12+ * + 8+ [M + 12H ] [M + 8H ] replacing the UV [M + 2DHB + Na+ + 7H+]8+ [M + 13H+]13+ * * * [M + 14H+]14+ absorbing matrix Absolute Abundance Absolute * [M + 7H+]7+ (DHB) with either 0 500 700 900 1100 1300 formic or acetic m/z Figure 5.4: liq-MALDESI-LTQ-FT-ICR mass spectra from 5 μMsolutions acid. Observed of A) bradykinin and B) melittin and C) 50 μM solution of ubiquitin mixed with DHB. Without ESI post-ionization, sample target biased at 3 kV. ion abundances
87 for the acidified samples were equivalent to samples prepared with DHB, indicating that ionization can occur without the addition of UV absorbing MALDI matrix (data
not shown).
The volume desorbed from the 0.5 μL precursor droplet (analyte + matrix in
50 % aqueous acetonitrile) for each mass spectrum can be estimated based on the time required to desorb/evaporate the droplet to dryness (~ 30 seconds) on the surface at room temperature (~ 25oC). An approximation can be made for the
amount of analyte removed per mass spectrum utilizing several basic assumptions
(e.g., desorption rate, constant concentration). Assuming a constant rate of
desorption, approximately 17 nanoliters are desorbed per second. For a 5 μM
solution this corresponds to ~ 85 femtomoles desorbed and detected per spectrum
(1 second collection). Detectable FT-ICR signal was obtained from a 1 μM bradykinin + DHB solution (S/N = 6.8, data not shown); using the same desorption rate this corresponds to ~ 17 femtomoles desorbed and detected per spectrum, equivalent to electrospray ionization of a 1 μM solution at a flow rate 1 μL/min with a
1 second collection.
The liq-MALDESI (without ESI post-ionization) ionization mechanism is hypothesized to result from the generation of excess protons in the bulk solution by oxidation of water at the sample target interface, which is supported by the data in
Figure 5.3B. The excess protons are electrostatically repulsed by the high potential at the sample target as well as from other protons, resulting in a high charge density
88 at the droplet surface, similar to the high charge density found at the surface of electrosprayed droplets.19 The UV laser energy is absorbed by organic matrix (DHB) dissolved in the droplet, resulting in an eruption of small highly-charged droplets
(offspring droplets) into the space above the macroscopic droplet, which then undergo an ESI-like desorption and ionization process generating multiply-charged ions en route to the mass spectrometer.
5.3.5 liq-MALDESI-LTQ-FT-ICR Electrochemical Ionization
Detailed examination of the liq-MALDESI-FT-ICR mass spectra of melittin revealed a small peak in the DHB adduct that corresponds to the triply-charged doubly adducted radical cation (data not shown). This unexpected result led to the
investigation of the
.+ 2268 M Rubrene electrochemical ionization
potential of this technique using a
rubrene, a molecule used by Van
Berkel’s group, with
demonstrated ability to form a Absolute Abundance
stable radical cation by
0 electrochemical oxidation.23 500 600 700 m/z Figure 5.5: liq-MALDESI-FT-ICR mass spectrum Rubrene dissolved in from 50 μM rubrene without ESI post-ionization, sample target biased at 3 kV. dichloromethane was diluted with
89 acetonitrile to give a 50 μM final concentration in 25 % dichloromethane 75 %
acetonitrile.
Liquid sample droplets (1.0 μL) were deposited onto the sample target biased at 3 kV for immediate (1 – 15 seconds, due to increased evaporation rate) laser desorption at a laser power of 50 μJ and 50 Hz repetition rate. The liq-MALDESI-FT-
ICR mass spectrum of rubrene is shown in Figure 5.5. The singly-charged radical cation was detected and determined by exact mass. The presence of the radical cation demonstrates the potential of this technique for ionization of electrochemically active molecules via oxidation.
5.4 Conclusions
liq-MALDESI is a sensitive atmospheric pressure ionization technique used to generate multiply-charged ions from a liquid sample drop biased at high potential.
The relatively easy sample preparation and sample accessibility during analysis are key features demonstrated by liq-MALDESI. Laser desorption from the liquid droplet eliminates the need for electrosprayed solvent for fast and sensitive analysis. The surface tension is attenuated by the applied voltage and may play a key role in desorption and ionization using liq-MALDESI. The generation of multiply-charged ions by liq-MALDESI results in increased top-down efficiency and when coupled to high resolving power high mass accuracy FT-ICR mass spectrometry is an excellent platform for identification and characterization of biological molecules. liq-MALDESI
90 also offers the advantage of eliminating carryover or cross-contamination between samples as each drop is deposited separately on a new portion of the sample target for analysis. We are currently investigating electronically addressable sample targets
(e.g., patterned surfaces) for independent control over applied potential and coupling to online HPLC for analysis of complex biological samples (e.g., biological fluids).
Furthermore, we are studying liq-MALDESI as an ionization method for electrochemically active molecules not normally ionizable using ESI.23, 24
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21. Grahame, D. C., The Electrical Double Layer and the Theory of Electrocapillarity. Chemical Reviews 1947, 41, 441-501.
22. Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C., Journal of the American Society for Mass Spectrometry 2008, submitted.
23. Van Berkel, G. J.; Zhou, F. M., Characterization of an Electrospray Ion- Source as a Controlled-Current Electrolytic Cell. Analytical Chemistry 1995, 67, 2916-2923.
24. Blades, A. T.; Ikonomou, M. G.; Kebarle, P., Mechanism of Electrospray Mass-Spectrometry - Electrospray as an Electrolysis Cell. Analytical Chemistry 1991, 63, 2109-2114.
94 Chapter 6
Intact and Top-Down Characterization of Biomolecules and Direct Analysis using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Coupled to FT-ICR Mass Spectrometry
6.1 Introduction
The recent growth in the development of hybrid ambient direct analysis techniques such as laser desorption atmospheric pressure chemical ionization (LD-
APCI)1, direct analysis in real time (DART)2, desorption electrospray ionization
(DESI)3, atmospheric-pressure solids analysis probe (ASAP)4, electrospray-assisted
laser desorption ionization (ELDI)5, 6, matrix-assisted laser desorption electrospray
ionization (MALDESI)7, 8, laser ablation electrospray ionization (LAESI)9 and infrared laser-assisted desorption electrospray ionization (IR-LADESI)10 have rapidly
expanded the analytical space accessible using mass spectrometry. LAESI and IR-
LADESI were introduced around the same time and are in principle the same
ionization method that may be better described as IR-MALDESI whereas
endogenous water or sacrificial analyte serves as the matrix for infrared matrix-
assisted laser desorption followed by ESI postionization. All of the techniques listed
above achieve ionization at atmospheric pressure with minimal sample preparation,
reducing sample handling and preparation times. Earlier work has reported the
ionization of biomolecules from liquids held in vacuo using IR radiation. 11-13
MALDESI and other similar laser desorption with ESI postionization techniques
95 generate multiply-charged ions and have the added advantage of increased top-
down fragmentation efficiency; thereby enhancing sequencing capabilities which is
invaluable for the identification and characterization of biological molecules using a
variety of fragmentation methodologies including CID, ETD, ECD, SORI and
IRMPD.14-18 The detection of multiply-charged ions in Fourier transform ion cyclotron
resonance (FT-ICR) and Orbitrap (LTQ-Orbi) yields increased resolving power and
increased mass accuracy (<3ppm)19, 20 due to the inverse relationship between the
frequency and m/z and √(m/z), respectively.21
Poor shot-to-shot reproducibility has been an issue with UV matrix-assisted
laser desorption techniques due to the inherent inhomogeneity of analyte/matrix co- crystallization.22 This inhomogeneity has been addressed by utilizing a liquid sample,
often mixed with glycerol for vacuum compatibility, thereby eliminating the co-
crystallization step in sample preparation.23, 24 In building upon this idea to improve
shot-to-shot reproducibility, liquid sample droplets have been utilized for atmospheric
pressure analysis using liquid matrix-assisted laser desorption electrospray
ionization (liq-MALDESI) resulting in increased shot-to-shot reproducibility.25
IR-MALDI has demonstrated desorption and ionization of large biomolecules utilizing both solid and liquid matrices26. It is well established that infrared laser
desorption generates many more neutral molecules than ions, thereby reducing
mass spectrometric detection sensitivity. Although IR-MALDI has demonstrated
practical utility for analysis of complex samples, the desorption and ionization
96 mechanisms are not well understood. It has been proposed that water molecules of hydration associated with the analyte (i.e. proteins) as well as the analyte itself absorbs infrared laser irradiation resulting in O-H, C-H and N-H bond stretching which result in the desorption of analyte as well as serve as a possible source of protons for ionization.27-29 The surrounding solvent may also act to isolate the analyte reducing cluster formation and collisionally cool the desorbed molecules, thereby reducing fragmentation.
The advantages of IR laser desorption stem from the capability of desorption and ionization directly from samples without the need for adding exogenous matrix.27, 28, 30 This combined with the deep penetration of infrared laser ablation results in ablation of large amounts of material, the majority of which are neutral molecules, makes infrared an ideal wavelength range for laser desorption using the
MALDESI platform.
Demonstrated herein we have integrated an IR laser (2.94 μm) into the existing versatile MALDESI ionization source.31 IR laser desorption from liquid- and solid-state samples followed with ESI postionization at atmospheric pressure generates multiply-charged peptide and protein ions. High mass accuracy for intact and top-down characterization of biomolecules (i.e., myoglobin, 17 kDa) is demonstrated. Direct analysis of commercially produced milk without sample preparation and farm raised eggs is demonstrated using both UV (349 nm) and IR
(2.94 μm) laser desorption. O-linked glycans cleaved from mucin32-34 were
97 sequenced from liquid-state samples using IR-MALDESI. Importantly, proteins,
carbohydrates and lipids were detected with minimal sample preparation using IR-
MALDESI coupled to FT-ICR mass spectrometry.
6.2 Experimental
6.2.1 Materials
Substance P, somatostatin, laminin, human angiotensin I, honey bee melittin, porcine glucagon, bovine ubiquitin, bovine cytochrome c, equine myoglobin, succinic
acid and glycerol were purchased from Sigma-Aldrich (St. Louis, MO) and used as
received. Bovine milk (2 %) was purchased from a local grocery store and used
without further preparation. Farm raised chicken eggs were prepared as indicated.
Porcine mucin was purchased from Sigma-Aldrich (St. Louis, MO) from which O-
linked glycans were cleaved from proteins using reductive β-elimination and
purification as previously reported.32-34 The specifics for the method employed are
detailed in several recent publications by our group.32, 34, 35 O-linked glycan samples
were from solid-phase extraction elution 1 (10 % acetonitrile) and used without
further preparation. HPLC grade acetonitrile, methanol and high purity water were
purchased from Burdick and Jackson (Muskegon, MI).
98 6.2.2 IR- and UV-MALDESI Mass Spectrometry
An erbium doped yttrium aluminum garnet (ER-YAG) infrared laser (IR)
(Bioptic Lasersysteme, Berlin, Germany) was mounted to the main working platform
of the previously described MALDESI source.31 The IR laser generated 2.8 mJ pulse
energy (measured at the laser) at 10 Hertz (Hz) repetition rate with a pulse width of
100 nanoseconds and emission wavelength of 2.94 micrometers (μm). The laser beam path was directed by two broadband gold coated Pyrex mirrors (Part#
10D20ER.4, Newport Corporation, Irvine, CA) and focused to a spot size of 200 μm using a calcium fluoride plano convex lens (Part # 47174, Edmund Optics,
Barrington, NJ) onto the stainless steel sample target (Part # 4333375, Applied
Biosystems, Foster City, CA). The laser fluence was calculated at 2.2 J/cm2.
The ultraviolet (UV) laser (Explorer, Newport Corporation, Irvine, CA) with an
emission wavelength of 349 nanometers (nm), computer controlled pulse energy (0-
120 μJ) and repetition rate (0-5000 Hz) was utilized with the same experimental set-
up as has been described previously.31 Briefly, two UV enhanced aluminum mirrors
(Part # 10D20AL.2, Newport Corporation, Irvine, CA) direct the laser beam which is focused to a spot size of 60 μm by a fused silica lens (Part # SPX017 + AR.10,
Newport Corporation, Irvine, CA) onto the sample target; the laser power was set to
50 μJ, measured internally. The laser fluence was calculated at 0.4 J/cm2.
The sample target was positioned in front of and below the extended ion transfer capillary of a hybrid LTQ-FT-ICR mass spectrometer. The LTQ-FT-ICR was
99 equipped with a 7 Tesla actively shielded superconducting magnet, the maximum ionization time was set to 1 second and resolving power set to 100,000 at 400 m/z.
Electrospray ionization was generated by infusing the ESI solution (50 % acetonitrile in water) through a 75 μm i.d. fused silica capillary (Polymicro Technologies,
Phoenix, AZ) connected with a stainless steel union (part # ZU1XC, VICI, Houston,
TX) to a 30 μm fused silica tapered Picotip (New Objective Inc., Woburn, MA) at a flow rate of 400-800 nL/min. using a syringe pump (Harvard Apparatus, Holliston,
MA). The stainless steel union connecting the transfer capillary to the ESI emitter tip was biased at 3 kilovolts (kV). All experiments were performed at ambient temperature and pressure. Top-down data analysis was processed manually by obtaining precursor m/z values from the MS/MS header and importing the MS/MS
values into ProSightPC (Thermo Electron, Waltham, MA) using a THRASH36 algorithm to determine the monoisotopic peak of the analyte signal as detailed in a recent publication by our group.37
6.3 Results and Discussion
6.3.1 Solid-State IR-MALDESI-FT-ICR
An aqueous solution of bovine cytochrome c was mixed 1:1 (v:v) with succinic
acid (47 mg/mL) in 50 % acetonitrile to give a final concentration of 200 μM. The
sample solution was deposited (0.8 μL) onto a stainless steel sample target and
actively dried under a cool stream of air.38 The sample target was installed onto the
100 MALDESI platform equipped with an infrared laser for IR-MALDESI analysis. The IR laser was manually actuated while 50 % acetonitrile in water was electrosprayed at 3 kV and a flow rate of 800 nL/min.; the sample target was biased at 500 volts (V). A schematic of solid-state IR-MALDESI and representative mass spectrum of cytochrome c are shown in Figure 6.1. Each single acquisition mass spectrum demonstrated high resolving power multiply-charged ions of cytochrome c for accurate mass determination and protein identification. The average charge state
[M + 9H+]9+ MS Inlet Capillary Mid-IR Laser 10584 + + + + + + + + + + + + + + + + + + + + ++ + + + + + 8+ + + + 10+ [M + 8H ] + + + + + + + + + [M + 10H ] + + + + + + + + + + + + 11+ + + + ESI Emitter [M + 11H ] + + + + + +40 V + +3000 V Cytochrome C [M + 12H+]12+ + [M + 7H+]7+ Succinic Acid Abundance Absolute +500 V 0 800 1400 2000 Figure 6.1 Schematic of solid-state IR-MALDESI with ESI postionizationm/z and representative mass spectrum of bovine cytochrome c mixed with succinic acid.
(ACS) for cytochrome c was calculated using the following equation (ACS =
(∑(charge state of each peak X abundance of each peak)) / (total abundance of
peaks)) and found to be (ACS = 9.41). Similar high resolving power multiply-charged
mass spectra were obtained from aqueous solutions of angiotensin I, somatostatin,
laminin, melittin, glucagon, ubiquitin and myoglobin, each mixed with succinic acid
and actively dried prior to analysis (data not shown); demonstrating desorption and
101 ionization over a broad peptide and protein molecular weight range (1.2-17 kDa),
similar to data previously demonstrated by UV-MALDESI.
Control experiments were performed by laser desorption of the same solid
samples with the ESI emitter removed to determine if ionization occurs via
atmospheric pressure IR-MALDI. All control experiments resulted in no measurable
analyte signal without ESI postionization.
In a separate experiment, the ESI emitter was removed and the recently
introduced remote analyte sampling, transport and ionization relay (RASTIR) was
installed.39 The RASTIR device serves to transport the neutral laser desorption
products by induced vacuum to the ionization region positioned directly in front of the mass spectrometer inlet capillary. This device separates in space the complex electrical fields of the sample target and the ESI emitter, enabling independent
optimization of each component. The electrospray emitter of the RASTIR device was
biased at 3 kV while the RASTIR body and the sample target were allowed to float
(not biased or grounded). Cytochrome c and myoglobin, each mixed with succinic
acid and actively dried, were subjected to IR laser desorption in close proximity to
the collection tube of the RASTIR device. The laser desorption products were
transported into the ionization region of the RASTIR device and subsequently
ionized, multiply-charged ions of cytochrome c and myoglobin, similar to those
obtained without the RASTIR device were detected (data not shown).
102 The ionization mechanism we propose is similar to that proposed for solid- state UV-MALDESI;7 laser desorbed neutral molecules or particles are absorbed into the electrosprayed charged solvent droplets which then undergo an ESI-like desorption and ionization process generating multiply-charged ions. We base our hypothesis on the similarity of the observed charge states to those obtained by ESI and the absence of ion signal with the ESI emitter removed, eliminating an AP-
MALDI mechanism. The RASTIR-IR-MALDESI experiments demonstrate remote electrospray postionization of laser desorption products and can therefore rule out a
DESI-like mechanism due to separation of the desorption event from the ionization event in both time and space as well as eliminating the possibility of laser desorption from a charged liquid droplet from the surface (the sample target is not biased and the ESI emitter is located remote from the laser desorption region).
This initial work is the first demonstration of RASTIR combined with IR-
MALDESI for the sole purpose of separating the desorption and ionization regions of
MALDESI and may be invaluable in the characterization of the ionization mechanism. The complex electric fields of the desorption and ionization regions due to the ESI emitter and sample target voltages in MALDESI can be avoided by implementing the RASTIR device, thereby allowing independent control of the ESI and sample target voltages for optimal desorption and ionization.
103 6.3.2 Liquid-State IR-MALDESI-FT-ICR
Aqueous samples of cytochrome c containing 10 % glycerol were deposited
(1.5 μL) and analyzed without drying by IR-MALDESI with ESI postionization. A
schematic of liquid-state IR-MALDESI and representative mass spectrum of
cytochrome c is shown in Figure 6.2. The sample target voltage (500 V), ESI
voltage (3 kV) and ESI flow rate (800 nL/min.) were the same as used in solid-state analysis. The liquid sample drop yielded signal only while the sample remained liquid on the surface (~1 minute), no signal was obtained after the droplet dried on the surface. Similar high ion abundances were observed, which indicates the
[M + 9H+]9+ 10215 [M + 10H+]10+ MS Inlet Capillary Mid-IR laser + + 11+ + + + + + [M + 11H ] ++ + + + + + + + + 12+ ++ ++ + + [M + 12H ] + 8+ + + + + + [M + 8H ] ++ + + + + + + + + + + + ESI Emitter + + + [M + 13H+]13+ + + + +3000 V + + +40 V [M + 14H+]14+ + + + + Cytochrome C + + + + + 15+ + [M + 15H ] + 7+ +500 V 10 % glycerol Absolute Abundance [M + 7H ] 0 500 1200 1800 m/z Figure 6.2 Schematic of liquid-state IR-MALDESI with ESI postionization and representative mass spectrum of bovine cytochrome c mixed with 10 % glycerol.
versatility of desorption and ionization from both solid-state and liquid-state samples
using this technique. The average charge state (ACS) of cytochrome c desorbed
from the liquid-state was calculated using the same equation as for solid-state
cytochrome c (vide supra) and found to be ACS =10.43. The average charge state
for liquid-state IR-MALDESI (10.4) was found to be 1 whole charge higher than the
104 average charge state for solid-state IR-MALDESI (9.4). One possible explanation for the increase in average charge state with liquid-state desorption is the absence of charge sequestering matrix molecules that are desorbed from the solid-state sample with every laser shot. Another explanation is that in the liquid-state analyte may be more efficiently desorbed (less energy is required to liberate the analyte from solution than from a solid lattice).
The potential for minimal sample preparation (i.e., no additional matrix) was explored by preparing analyte solutions, without the addition of the matrix glycerol.
IR-MALDESI analysis of substance p, somatostatin, laminin, angiotensin I, melittin, glucagon, ubiquitin, cytochrome c and myoglobin demonstrated desorption and ionization via IR-MALDESI without addition of matrix, generating multiply-charged ions (data not shown).
Control experiments were performed using new droplets of the same analyte solutions (no matrix) with the ESI emitter removed and high potential (0-5000 V) applied directly to the sample target. Only the liquid-state IR-MALDESI mass spectrum of angiotensin I revealed analyte signal, yielding only the singly-charged species, the doubly-charged ion was not observed. The singly-charged ion indicates an atmospheric pressure MALDI ionization process from the liquid droplet. None of the other peptides or proteins used in these control experiments demonstrated measurable analyte signal without ESI postionization.
105 6.3.3 Intact and Top-Down Analysis using IR-MALDESI-FT-ICR
Equine myoglobin mixed with succinic acid was deposited onto the sample target and dried with a cool stream of air. The sample was desorbed and ionized by
IR-MALDESI and sampled by the mass spectrometer. The most abundant charge state, [M + 15H+]15+ was isolated for collision induced dissociation, the precursor and fragment ions were detected in the ICR cell. The expanded region of the precursor is
y122 b1 G L S D G E W Q Q V L N V W G K V E A D I A G H G Q E V L I R L F T G H P E T L E K F D K F K H L K T E A E M K A S E D L K y91 b32 K H G T V V L T A L G G I L K K K G H H E A E L K P L A Q S H y60 b63 A T K H K I P I K Y L E F I S D A I I H V L H S K H P G D F G y29 b94 A D A Q G A M T K A L E L F R N D I A A K Y K E L G F Q G y1 b123 MMA = 1.98 ppm [M+15H]15+ [M+15H]15+
MMA = 0.95 – 5.63 ppm
MS/MS 14+ y151
14+ y149
y 14+ 4+ 4+ 9+ 147 y34 y35 y90 5+ y57
1130 1132.5 900 1000 1100 1200 1300 m/z m/z Figure 6.3 Top-down sequencing of equine myoglobin using solid-state IR-MALDESI with ESI postionization.
shown in the lower left of Figure 6.3 with the MS/MS spectrum with the precursor and fragment ions shown in the lower right of Figure 6.3. The amino acid sequence with the identified tandem MS fragments indicated is shown in the top of Figure 6.3.
106 The mass measurement accuracy for the precursor (MMA = 1.98 parts-per-million) and fragment ions (MMA = 0.95-5.63 parts-per-million) were calculated manually.
6.3.4 Direct Analysis using UV- and IR-MALDESI
Direct analysis via UV and IR laser desorption with ESI postionization is demonstrated for bovine milk and chicken egg yolk as shown in Figure 6.4. The milk was used without preparation and analyzed in the liquid-state. The UV- and IR-
[M + Na+]1+ [M + K+]1+ 897.7315 1150 26608 Lactose Lipids UV laser desorption a,b [2M + Na+]1+ 923.7472 869.7002 [2M + K+]1+ Abs. Abundance Abs. Abundance Abs. 0 0 (a) (b) [M + Na+]1+
+ 1+ 895.7151 [M + K ] 15530 17630 Lactose Lipids
IR laser [2M + Na+]1+ 798.5413 desorption [2M + K+]1+ 923.7474 1518.1479 c,d 1546.1623 Abs. Abundance Abs. Abs. Abundance Abs. 0 0 (c) 200 800 1400 (d) 200 1000 1800 m/z m/z Figure 6.4 UV Liquid-state MALDESI mass spectra of a) bovine milk with no sample preparation using and b) egg yolk with added DHB matrix. IR Liquid-state MALDESI mass spectra of c) bovine milk and d) egg yolk, both without sample preparation.
MALDESI mass spectra of bovine milk are shown in Figures 6.4A and 6.4B, respectively, both with ESI postionization. The main peaks observed in the in the
107 mass spectra were identified as the sodium and potassium adducts of lactose as
well as the sodium and potassium adducted dimers of lactose. The IR-MALDESI
mass spectrum has several other unidentified peaks, indicating desorption and
ionization of other components of the milk sample not observed in the UV-MALDESI
mass spectrum. The absolute abundance was substantially higher when using the
IR laser as compared to UV laser desorption. The increased abundance may be
attributed to the increased number of neutral molecules desorbed by IR laser
ablation available for ESI postionization.
The UV- and IR-MALDESI mass spectrum of chicken egg yolk (mixed with
DHB for UV laser desorption) are shown in Figures 6.4C and 6.4D, respectively.
The larger ion abundance observed for UV-MALDESI is likely due to incorporation of the UV absorbing matrix (DHB) which was required to obtain appreciable signal. The unidentified main peaks of the resulting mass spectra were determined to be lipids based on their mass excesses. The mass excess (defined by IUPAC) is calculated by the difference between the nominal mass and the exact mass, the mass excess was determined for each of the main peaks in the UV- and IR-MALDESI mass spectrum of egg yolk. The mass excesses were plotted onto a heat map of the mass excess of tryptic peptides versus the monoisotopic mass which demonstrates the forbidden zones for tryptic peptides.40 The mass excess of the peaks fell outside of
the range normally occupied by tryptic peptides indicating that the observed ions are probably not peptides or proteins. The mass excess of several common lipids were
108 plotted onto the same heat map which fell into the same range, indicating that these
are likely lipids. A small group of higher mass peaks were observed in the IR-
MALDESI mass spectra of egg yolk (Figure 4D) which were not observed in the UV-
MALDESI mass spectra of egg yolk (Figure 4C). These higher mass ions may indicate more efficient desorption of higher mass molecules from complex samples using IR-MALDESI as compared to UV-MALDESI.
6.3.5 Carbohydrate Analysis using IR-MALDESI
Liquid-state samples (0.8 μL) of elution 1 from the β-elimination/solid-phase
extraction process were deposited onto the sample target for direct IR-MALDESI
analysis with ESI postionization. The IR-MALDESI mass spectrum is shown in
Figure 6.5. The circles denote identified sodium adducted O-linked glycans. The
diamonds denote identified protonated O-linked glycans. The identification of the
glycans was verified by exact mass and tandem MS. A representative glycan
([4HexNAc:2Hex + Na+]1+, m/z = 1179.4386) was isolated and subsequently
fragmented with collision induced dissociation and is shown in the inset of Figure
6.5.
109 The fragment ions correspond to losses of HexNAc, HexNAc:Hex, and
2HexNac:Hex; the mass measurement accuracy associated with this precursor peak
was -0.34 parts-per-million. The IR-MALDESI mass spectrum was compared with
MALDI-FT-ICR and -HexNAc DESI-FT-ICR mass [4HexNAc:2Hex + Na+]1+ spectra of the same -HexNAc:Hex
-2HexNAc:Hex elution as has been
reported previously.35 400 800 1200 m/z The IR-MALDESI mass Sodium adducted
Protonated spectra yielded many of the same peaks
identified using MALDI 400 600 800 1000 1200 1400 m/z and DESI. MALDESI Figure 6.5 Liquid-state IR-MALDESI of O-linked glycans cleaved from mucin using reductive β-elimination chemistry. Circles denote avoids fragmentation sodium adducted glycans, diamonds denote protonated glycans. Inset shows MS/MS sequencing of glycan with m/z = 1179.4386. commonly encountered when using MALDI and when coupled with a hybrid mass analyzer provides top- down sequence information and high mass accuracy. MALDESI is also much more sensitive, requiring approximately ¼ the amount of material required for DESI as demonstrated in our laboratory35 for the analysis of carbohydrates.
110 6.4 Conclusions
IR-MALDESI is an atmospheric pressure ionization source for analysis of biomolecules with minimal sample preparation requiring no pretreatment necessary for vacuum sensitive samples (i.e., tissues). The liquid- or solid-state sample remains accessible during analysis allowing flexibility in sample preparation (i.e., solid or liquid) as well as with or without added matrix and the type of matrix used, demonstrating the inherent flexibility and versatility for biomolecular analysis.
IR-MALDESI intact and top-down proteomic strategies have been employed for identification and characterization of biological molecules including proteins and glycans, which are invaluable for analysis of complex samples (e.g., tissues, bio- fluids). IR-MALDESI direct analysis without addition of an exogenous matrix has the
unique potential for direct analysis without sample preparation for generation of
multiply-charged ions from biological samples by utilizing the endogenous matrix
(solvent or sacrificial analyte). Furthermore the inherent versatility of the MALDESI ionization source is demonstrated to include both UV and IR laser desorption with
ESI postionization for the generation of multiply-charged ions and the potential application for other desorption and ionization techniques. Future studies include utilizing the RASTIR device to gain a clear understanding of the IR-MALDESI ionization mechanism which will enhance the capabilities of this technique as well as potentially elucidate other laser desorption/ionization mechanisms (i.e., MALDI).
111 6.5 References
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26. Overberg, A.; Karas, M.; Bahr, U.; Kaufmann, R.; Hillenkamp, F., Matrix- Assisted Infrared-Laser (2.94-Mu-M) Desorption Ionization Mass- Spectrometry of Large Biomolecules. Rapid Communications in Mass Spectrometry 1990, 4, 293-296.
27. Berkenkamp, S.; Karas, M.; Hillenkamp, F., Ice as a matrix for IR-matrix- assisted laser desorption/ionization: Mass spectra from a protein single crystal. Proceedings of the National Academy of Sciences of the United States of America 1996, 93, 7003-7007.
28. Little, M. W.; Laboy, J.; Murray, K. K., Wavelength dependence of soft infrared laser desorption and ionization. Journal of Physical Chemistry C 2007, 111, 1412-1416.
29. Menzel, C.; Dreisewerd, K.; Berkenkamp, S.; Hillenkamp, F., Mechanisms of energy deposition in infrared matrix-assisted laser desorption/ionization mass spectrometry. International Journal of Mass Spectrometry 2001, 207, 73-96.
114 30. Rousell, D. J.; Dutta, S. M.; Little, M. W.; Murray, K. K., Matrix-free infrared soft laser desorption/ionization. Journal of Mass Spectrometry 2004, 39, 1182-1189.
31. Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C., Construction of a Versatile High Precision Ambient Ionization Source for Direct Analysis and Imaging. Journal of the American Society for Mass Spectrometry 2008, 19, 1527-1534.
32. Williams, T. I.; Saggese, D. A.; Muddiman, D. C., Studying O-linked protein glycosylations in human plasma. Journal of Proteome Research 2008, 7, 2562-2568.
33. An, H. J.; Miyamoto, S.; Lancaster, K. S.; Kirmiz, C.; Li, B. S.; Lam, K. S.; Leiserowitz, G. S.; Lebrilla, C. B., Profiling of glycans in serum for the discovery of potential biomarkers for ovarian cancer. Journal of Proteome Research 2006, 5, 1626-1635.
34. Williams, T. I.; Saggese, D. A.; Toups, K. L.; Frahm, J. L.; An, H. J.; Li, B.; Lebrilla, C. B.; Muddiman, D. C., Investigations with O-linked protein glycosylations by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry. Journal of Mass Spectrometry 2008, 43, 1215-1223.
35. Bereman, M. S.; Williams, T. I.; Muddiman, D. C., Carbohydrate analysis by desorption electrospray ionization fourier transform ion cyclotron resonance mass spectrometry. Analytical Chemistry 2007, 79, 8812-8815.
36. Horn, D. M.; Zubarev, R. A.; McLafferty, F. W., Automated reduction and interpretation of high resolution electrospray mass spectra of large molecules. Journal of the American Society for Mass Spectrometry 2000, 11, 320-332.
37. Collier, T. S.; Hawkridge, A. M.; Georgianna, D. R.; Payne, G. A.; Muddiman, D. C., Top-down identification and quantification of stable isotope labeled proteins from Aspergillus flavus using online nano-flow reversed-phase liquid chromatography coupled to a LTQ-FTICR mass spectrometer. Analytical Chemistry 2008, 80, 4994-5001.
38. Williams, T. I.; Saggese, D. A.; Wilcox, R. J.; Martin, J. D.; Muddiman, D. C., Effect of matrix crystal structure on ion abundance of carbohydrates by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry. Rapid Communications in Mass Spectrometry 2007, 21, 807-811.
115 39. Dixon, R. B.; Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C., Ambient aerodynamic ionization source for remote analyte sampling and mass spectrometric analysis. Analytical Chemistry 2008, 80, 5266-5271.
40. Frahm, J. L.; Howard, B. E.; Heber, S.; Muddiman, D. C., Accessible proteomics space and its implications for peak capacity for zero-, one- and two-dimensional separations coupled with FT-ICR and TOF mass spectrometry. Journal of Mass Spectrometry 2006, 41, 281-288.
116 Chapter 7
Atmospheric Pressure Infrared (10.6 µm) Laser Desorption Electrospray Ionization (IR-LDESI) Coupled to a LTQ-FT-ICR Mass Spectrometer
7.1 Introduction
Soft ionization sources such as matrix-assisted laser desorption ionization
(MALDI)1, 2 and electrospray ionization (ESI)3 have increased both the quantity and quality of information obtainable from biological samples. MALDI has demonstrated
a high tolerance to ion suppressing agents (i.e., salts) common to biological matrices
while providing data across a wide molecular weight range. However, MALDI is not
amenable to sequencing of large molecules using the top-down approach due to its
limitation of generation of primarily singly-charged ions. ESI has demonstrated utility
using both the bottom-up and top-down approach for identification and
characterization peptides and proteins and is of particular value in sequencing of
large biomolecules due to multiple-charging inherent to the ESI process. The
information gained from the intact and top-down approach is invaluable for the
characterization and identification of intact biomolecules; however, extensive sample
preparation (e.g., HPLC) is often required for complex biological samples prior to analysis.
Recent innovations in ionization source development such as desorption electrospray ionization (DESI),4 direct analysis in real time (DART),5 atmospheric
pressure solids analysis probe (ASAP),6 electrospray-assisted laser desorption
117 ionization (ELDI),7-10 matrix-assisted laser desorption electrospray ionization
(MALDESI),11-15 laser ablation electrospray ionization (LAESI),16 infrared laser-
assisted desorption electrospray ionization (IR LADESI)17 and radio frequency
acoustic desorption ionization (RADIO)18 have continued to expand the capabilities
of mass spectrometry by reducing sample preparation required prior to atmospheric
pressure analysis. These “hybrid” ionization techniques share the common theme,
atmospheric pressure ionization with minimal sample preparation. Of particular
interest is laser desorption with ESI postionization (i.e., MALDESI), whereby minimal sample preparation is required due to the separation of laser ablation (UV or IR) of the complex sample from ESI postionization for the generation of multiply-charged ions. This technique enables one to capitalize on minimal sample preparation while preserving the intact and top-down sequencing capabilities for characterization and identification of biological molecules.12
In vacuo infrared laser desorption has been demonstrated previously by
Dreisewerd et al. using infrared laser desorption ionization (IR-LDI)19 of native
tissues with minimal sample preparation; however, generating primarily singly-
charged ions which severely limits top-down characterization capabilities. Konig et
al. demonstrated atmospheric pressure laser desorption/ionization which they
termed AP-MALDI,20 in which a dilute acid is added to the liquid sample mixed with
glycerol matrix deposited onto a biased target; yielding multiply-charged ions similar
to those observed in ESI and was speculated to involve ESI in the ionization
118 process. Recently, in our laboratory we have demonstrated multiple-charging from
solid- and liquid-state samples mixed with matrices at atmospheric pressure utilizing an IR laser (2.94 µm) for analysis of biological molecules including peptides, proteins, carbohydrates and lipids, which we termed IR-MALDESI.15
In previous experiments matrix or additives have been necessary for laser
desorption and postionization with ESI for typical MALDESI experiments.11-13 We have theorized that ionization occurs by a mechanism in which the laser desorbed neutrals are postionized by partitioning into ESI droplets. In other experiments from liquid droplets on a highly charged sample target,14 we theorized that charged
droplets are desorbed from the sample droplet which then undergo an ESI-like
desorption and ionization process. In either case the ionization mechanism appears
to follow an ESI-like desorption and ionization process producing multiply-charged
ions.
In the setup described herein a CO2 laser (10.6 µm) was incorporated into a
versatile atmospheric pressure ionization source13 for desorption and ionization of
proteins ranging from 8.6 to 17 kDa. Multiply-charged ions were generated from
liquid-state samples without the addition of matrix in a technique which we have
termed infrared laser desorption electrospray ionization (IR-LDESI).
119 7.2 Experimental
7.2.1 Materials
Formic acid, bovine ubiquitin, bovine myoglobin and bovine cytochrome c
were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received.
HPLC grade acetonitrile and high purity water were purchased from Burdick and
Jackson (Muskegon, MI, USA).
7.2.2 IR-LDESI Source and LTQ Mass Spectrometer
The versatile atmospheric pressure ionization source has been described
13 previously and is used here with the addition of a Synrad CO2 laser (J48-2-6541,
Synrad Inc., Mukilteo, WA, USA). The CO2 laser with 10.6 µm emission wavelength
and 5 kHz repetition rate was used with <10% (<100 µJ) laser power modulated
using a universal controller (UC-2000, Synrad Inc., Mukilteo, WA, USA). The CO2 laser (equipped with a co-aligned diode pointer laser) was mounted to the main working platform of the atmospheric pressure ionization source. The infrared laser beam was directed and focused using two broadband metallic mirrors (10D20ER.4,
Newport Corporation, Irvine, CA, USA) and one ZnSe Plano convex focusing lens
(NT48-020, Edmund Optics, Barrington, NJ, USA) onto the stainless steel sample target to a laser spot size of approximately 200 µm.
The sample target was positioned in front of and below the extended ion transfer capillary inlet of a hybrid LTQ-FT-ICR mass spectrometer (Thermo Fisher
120 Scientific, San Jose, CA, USA) equipped with an actively-shielded 7 Tesla superconducting magnet. Electrospray ionization was accomplished by pumping solvent at 400 nL/min. using a syringe pump (PhD 2000, Harvard Apparatus,
Holliston, MA, USA) through 75 µm i.d. fused silica capillary attached to a 30 µm tapered fused silica PicoTip (New Objective Inc., Woburn, MA, USA) using a stainless steel union (ZU1XC, VICI, Houston, TX, USA). The sample target was biased at 0 to 4000 volts using the on-board power supply (Analytica of Branford
Inc., Branford, CT, USA).
7.3 Results and Discussion
Droplets (1 µL) of a 10 µM solution of bovine ubiquitin and 25 µM solution of bovine myoglobin were prepared in 50% acetonitrile with 0.1% formic acid and each deposited onto the stainless steel sample target for immediate laser desorption while
electrospraying 50% acetonitrile with 0.1% formic acid. The IR-LDESI source configuration with ESI postionization is depicted in the diagram in Figure 7.1A. The laser power and applied sample target voltage were varied from 0% to 25% (0 to
300 µJ) and 0 V to 4000 V, respectively, to determine the optimal desorption and ionization conditions: maximum ion abundance was found utilizing 6% laser power
(30 µJ) with 800 volts applied to the sample target (data not shown). Figure 7.1B and 7.1C show representative IR-LDESI-FT-ICR mass spectra from a 10 µM
121 solution of bovine ubiquitin and a 25 µM solution of bovine myoglobin respectively, each desorbed from a 1 µL liquid droplet using a CO2 laser with ESI postionization.
MS Inlet Capillary + IR laser (10.6 μm) + + A + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ESI Emitter + + + + +2600 V +35 V + + Aqueous + + + Analyte
+800 V
[M + 10H+]10+ [M + 19H+]19+ B C [M + 20H+]20+ 2038 3389 [M + 18H+]18+ [M + 11H+]11+ [M + 9H+]9+ [M + 8H+]8+ [M + 17H+]17+ + 7+ [M + 7H ] [M + 16H+]16+ [M + 21H+]21+ [M + 12H+]12+ [M + 15H+]15+ [M + 14H+]14+ Absolute Abundance Absolute Abundance Absolute [M + 22H+]22+ [M + 13H+]13+ [M + 23H+]23+
0 0 600 1000 1400 600 1000 1400 m/z m/z Figure 7.1 A) IR-LDESI source configuration with infrared laser desorption from a liquid droplet with ESI postionization. IR-LDESI-FT-ICR mass spectra from a 1 µL droplet of B) 10 µM bovine ubiquitin and C) 25 µM bovine myoglobin in 50% acetonitrile with 0.1% formic acid with ESI postionization, sample target biased at 800 volts.
Control experiments were performed using the same sample solutions with the ESI emitter removed over a wide range applied target potentials (0 V to 4000 V) and laser powers (0% to 25%, 0 to 300 µJ); no analyte signal was observed with the
ESI emitter removed. Solutions of bovine ubiquitin and bovine myoglobin dissolved
122 in 100% water were also analyzed with IR-LDESI with ESI postionization; however,
no analyte signal was detected.
A 1 µL droplet of a 10 µM solution of cytochrome c prepared in 50%
acetonitrile with 0.1% formic acid was deposited onto the target for laser desorption
with ESI postionization. Surprisingly, no analyte signal was observed using identical
instrument parameters as those for ubiquitin and myoglobin. The laser power (0% to
25%, 0 to 300 µJ), ESI potential (1.2 kV to 4.0 kV), ESI flow rate (200 nL/min to 400
nL/min) and target potential (0 V to 4000 V) were all varied, with no resulting analyte
signal. The ESI emitter was removed and the potential applied to the sample target
varied (0 V to 4500 V), again surprisingly analyte signal was observed between 3250
V and 4500 V, with maximum ion abundance at 4000 volts (data not shown).
Once analyte signal was obtained the sample solution composition was
varied to improve the ion abundances; 100% water gave the most abundant analyte
A B [M + 12H+]12+ [M + 11H+]11+ 348 [M + 13H+]13+ MS Inlet Capillary + IR laser (10.6 μm) + 14+ + + + [M + 14H ] + + + + + + + + + + + + + + + + + [M + 10H+]10+ + + + + + + + + + + 15+ + + [M + 15H ] + + + + 16+ + + + + [M + 16H ] + + + [M + 9H+]9+ + + 17+ + + [M + 17H ] +35 V + Aqueous + [M + 8H+]8+ + + Analyte Abundance Absolute + + 7+ [M + 18H+]18+ [M + 7H ] +4000 V 0 600 1200 1800 m/z Figure 7.2 A) IR-LDESI configuration with infrared laser desorption from a liquid droplet without ESI postionization. B) IR-LDESI mass spectrum from a 1 µL droplet of 10 µM cytochrome c in 100% water without ESI postionization, sample target biased at 4 kV.
123 signal. The IR- LDESI source configuration without ESI postionization is depicted in
the diagram in Figure 7.2A. A representative IR-LDESI mass spectrum from a 1 µL
droplet of a 10 µM solution of cytochrome c in water deposited onto the stainless steel sample target biased at 4000 volts without ESI postionization is shown in
Figure 7.2B.
The result of ionization occurring only without ESI postionization for cytochrome c is intriguing; however, one possible theory is that cytochrome c may be charged upon desorption due to the 20 basic amino acids from the solution which would prevent it from partitioning into the highly-charged electrospray droplets. No ions were observed for any other samples without ESI postionization, thus suggesting this phenomenon is attributed specifically to IR-LDESI of cytochrome c.
7.4 Conclusions
IR-LDESI of proteins from aqueous solution is demonstrated utilizing a CO2 laser (10.6 µm) without the addition of a matrix. Multiply-charged ions of ubiquitin and myoglobin are detected with ESI postionization and for cytochrome c without
ESI postionization. Although the ionization mechanism is not well understood the experiments demonstrated herein for liquid droplets with ESI postionization appears to follow our previously stated hypothesis that laser desorbed neutrals interact with charged ESI droplets which undergo an ESI-like desorption and ionization process.
Laser desorption from liquid droplets without ESI postionization may also occur
124 through an ESI-like desorption and ionization process, whereby desorbed analyte contained within charged droplets undergo an ESI-like desorption and ionization process. These observation based hypotheses require further investigation and is the focus of ongoing research in our laboratory. IR-LDESI is ideal for analysis of water rich, vacuum sensitive samples in which high resolving power and high mass accuracy information is desired. To date, we have demonstrated the different ionization modalities including the use of UV wavelengths (337 and 349 nm), IR wavelengths (2.94 and 10.6 μm), and acoustical methods to produce multiply- charged ions from solid and/or liquid samples.11, 12, 14, 15, 18 Moreover, we accomplished this in some cases without the use of matrix. Combined, these new ionization methods provide fertile ground for future studies both fundamentally as well as to address emerging new applications.
125 7.5 References
1. Karas, M.; Hillenkamp, F., Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10000 Daltons. Analytical Chemistry 1988, 60, 2299-2301.
2. Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T., Protein and Polymer Analysis up to m/z 100 000 by Laser Ionization Time-of-Flight Mass Spectrometry. Rapid Communications in Mass Spectrometry 1988, 2, 151- 153.
3. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., Electrospray Ionization for Mass-Spectrometry of Large Biomolecules. Science 1989, 246, 64-71.
4. Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G., Mass spectrometry sampling under ambient conditions with desorption electrospray ionization. Science 2004, 306, 471-473.
5. Cody, R. B.; Laramee, J. A.; Durst, H. D., Versatile new ion source for the analysis of materials in open air under ambient conditions. Analytical Chemistry 2005, 77, 2297-2302.
6. McEwen, C. N.; McKay, R. G.; Larsen, B. S., Analysis of solids, liquids, and biological tissues using solids probe introduction at atmospheric pressure on commercial LC/MS instruments. Analytical Chemistry 2005, 77, 7826-7831.
7. Shiea, J.; Huang, M. Z.; Hsu, H. J.; Lee, C. Y.; Yuan, C. H.; Beech, I.; Sunner, J., Electrospray-assisted laser desorption/ionization mass spectrometry for direct ambient analysis of solids. Rapid Communications in Mass Spectrometry 2005, 19, 3701-3704.
8. Huang, M. Z.; Hsu, H. J.; Lee, J. Y.; Jeng, J.; Shiea, J., Direct Protein Detection from Biological Media through Electrospray-Assisted Laser Desorption Ionization/Mass Spectrometry. Journal of Proteome Research 2006, 5, 1107-1116.
9. Huang, M. Z.; Hsu, H. J.; Wu, C. I.; Lin, S. Y.; Ma, Y. L.; Cheng, T. L.; Shiea, J., Characterization of the chemical components on the surface of different solids with electrospray-assisted laser desorption ionization mass spectrometry. Rapid Communications in Mass Spectrometry 2007, 21, 1767- 1775.
126 10. Peng, I. X.; Shiea, J.; Loo, R. R. O.; Loo, J. A., Electrospray-assisted laser desorption/ionization and tandem mass spectrometry of peptides and proteins. Rapid Communications in Mass Spectrometry 2007, 21, 2541-2546.
11. Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C., Generation and detection of multiply-charged peptides and proteins by matrix-assisted laser desorption electrospray ionization (MALDESI) Fourier transform ion cyclotron resonance mass spectrometry. Journal of the American Society for Mass Spectrometry 2006, 17, 1712-1716.
12. Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C., Direct characterization of intact polypeptides by matrix-assisted laser desorption electrospray ionization quadrupole Fourier transform ion cyclotron resonance mass spectrometry. Rapid Communications in Mass Spectrometry 2007, 21, 1150- 1154.
13. Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C., Construction of a Versatile High Precision Ambient Ionization Source for Direct Analysis and Imaging. Journal of the American Society for Mass Spectrometry 2008, 19, 1527-1534.
14. Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C., Development and characterization of an ionization technique for analysis of biological macromolecules: Liquid matrix-assisted laser desorption electrospray ionization. Analytical Chemistry 2008, 80, 6773-6778.
15. Sampson, J. S.; Murray, K. K.; Muddiman, D. C., Intact and Top-Down Characterization of Biomolecules and Direct Analysis Using Infrared Matrix- Assisted Laser Desorption Electrospray Ionization Coupled to FT-ICR Mass Spectrometry. Journal of the American Society for Mass Spectrometry 2008, In Press.
16. Nemes, P.; Vertes, A., Laser ablation electrospray ionization for atmospheric pressure, in vivo, and imaging mass spectrometry. Analytical Chemistry 2007, 79, 8098-8106.
17. Rezenom, Y. H.; Dong, J.; Murray, K. K., Infrared laser-assisted desorption electrospray ionization mass spectrometry. Analyst 2008, 133, 226-232.
18. Dixon, R. B.; Sampson, J. S.; Muddiman, D. C., Generation of Multiply Charged Peptides and Proteins by Radio Frequency Acoustic Desorption and Ionization for Mass Spectrometric Detection. Journal of the American Society for Mass Spectrometry 2008, In Press.
127 19. Dreisewerd, K.; Draude, F.; Kruppe, S.; Rohlfing, A.; Berkenkamp, S.; Pohlentz, G., Molecular analysis of native tissue and whole oils by infrared laser mass spectrometry. Analytical Chemistry 2007, 79, 4514-4520.
20. Konig, S.; Kollas, O.; Dreisewerd, K., Generation of highly charged peptide and protein ions by atmospheric pressure matrix-assisted infrared laser desorption/ionization ion trap mass spectrometry. Anal Chem 2007, 79, 5484- 5488.
128 APPENDIX
129 APPENDIX A
Supplemental Figures
21 18 LTQ-FT MS inlet 15 31 Monitor 2 46 1 21 18 Laser
19 30 Computer Keyboard 20
53 6 Motion Control
27 Power Supply
50 24
Figure 4S1 Schematic of the MALDESI source by part number
Dried sample spot
Laser ablation path
Start End 2 mm
Figure 4S2 Ablation path for MALDESI whole spot analysis custom program
130
[M + 2H+]2+ 142002 A
Bradykinin
Absolute Abundance
[M + 1H+]1+
0 500 800 1200 m/z [M + 4H+]4+ 98348 B
Melittin
Absolute Abundance Absolute [M + 3H+]3+
[M + 5H+]5+
0 500 800 1200 m/z Figure 4S3 MALDESI FT-ICR mass spectrum of A) bradykinin and B) melittin both mixed with DHB
131
Myoglobin [M + 16H+]16+
+ 17+ [M + 17H ] + 15+ A 2851 [M + 15H ] [M + 18H+]18+
[M + 14H+]14+ [M + 19H+]19+ + 13+ [M + 13H ] [M + 20H+]20+
[M + 12H+]12+ Absolute Abundance [M + 11H+]11+ + 21+ [M + 21H ]
0
[M + 8H+]8+ 68132 [M + 6H+]6+ B Ubiquitin
[M + 9H+]9+ [M + 7H+]7+
[M + 10H+]10+
Absolute Abundance Absolute
[M + 11H+]11+
0 600 1100 1600 m/z Figure 4S4 Liquid MALDESI FT-ICR mass spectra of A) myoglobin and B) ubiquitin with ESI postionization
132 APPENDIX B
Glossary: Definition of terms and abbreviations italicized in dissertation
ACS average charge state
AE air ejector
AGC automatic gain control
APCI atmospheric pressure chemical ionization
AP-MALDI atmospheric pressure matrix-assisted laser desorption ionization
APPI atmospheric pressure photo ionization
ASAP atmospheric-pressure solids analysis probe
average charge state (ACS) calculated by the weighted average of the number of charges based on its abundance
bottom-up proteomics protein identification made from the accurate masses of an enzymatically or chemically cleaved protein and the MS/MS masses
CID collisionally induced dissociation
CCD charge coupled device
cyclotron frequency frequency at which ions spin around the center of the magnetic field inside the ICR cell
DART direct analysis in real time
DESI desorption electrospray ionization
DHB 2,5-dihydroxybenzoic acid
ECD electron capture dissociation
133 ELDI electrospray assisted laser desorption ionization
ESI electrospray ionization
ETD electron transfer dissociation
FD-ESI fused droplet electrospray ionization
FT-ICR Fourier transform ion cyclotron resonance
HPLC high performance liquid chromatography hybrid ionization an ionization source which utilizes properties from two or more existing ionization sources for which there exists a distinct benefit or attribute from each source hydrophobicity degree to which a peptide or protein is repelled by water; measured by the GRAVY (grand average of hydropathy) score based on the sums of the hydrophobicity of each component amino acid
ICR ion cyclotron resonance iodopeptides internal calibrant peptides derivatized with 3,5- diiodotyrosine ion suppression change in the abundance of ions due to less volatile compounds that changes the efficiency of droplet evaporation in ESI
IR infrared
IRMPD infrared multi-photon dissociation
IR-LDESI infrared laser desorption electrospray ionization
LAESI laser ablation electrospray ionization
LD laser desorption
Liquid-MALDESI liquid matrix-assisted laser desorption electrospray ionization
134
Lorentz force force exerted on a charged particle in an electromagnetic field
LTQ linear quadrupole ion trap mass spectrometer
LTQ-FT hybrid linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometer
LTQ-Orbi hybrid linear quadrupole ion trap Fourier transform mass spectrometer with electrostatic trapping cell
MALDESI matrix-assisted laser desorption electrospray ionization
MALDI matrix assisted laser desorption ionization
MMA mass measurement accuracy
MS mass spectrometry
MS/MS fragmentation mass spectrum of precursor ion m/z mass to charge ratio on an ion measured in mass spectrometry nanoESI nanoliter per minute flow rate ESI
PAGE polyacrylamide gel electrophoresis ppm parts-per-million
QFT-ICR quadrupole Fourier transform ion cyclotron resonance
RASTIR remote analyte sampling transport and ionization relay
RF radio frequency
RP resolving power
SA sinapinic acid
SIL stable isotope labeled
135
S/N signal to noise ratio
soft ionization ionization with little or no fragmentation
SORI sustained off resonance irradiation
space charge ion-ion repulsions in a spatially non-uniform electromagnetic field that shifts ICR frequencies due to ions experiencing variable internal and external fields
TOF time of flight mass spectrometer
top-down protein identification using the accurate masses of the intact protein and gas phase dissociation fragments (MS/MS)
Taylor cone cone-jet of charged particles emitted from conductive liquid at an applied potential where the electric field exerts a force in excess of the surface tension (i.e. ESI)
UV ultraviolet
136