Anal. Chem. 1996, 68, 873-882
Matrix-Enhanced Secondary Ion Mass Spectrometry: A Method for Molecular Analysis of Solid Surfaces Kuang Jen Wu* and Robert W. Odom
Charles Evans & Associates, Redwood City, California 94063
A new methodology, matrix-enhanced secondary ion mass spectrometry (ME-SIMS), is reported for the molecular analysis of biomaterials. The technique applies static secondary ion mass spectrometry (SSIMS) techniques to samples prepared in a solid organic matrix similar to sample preparations used in matrix-assisted laser desorption/ionization (MALDI). Molecular ions are observed in this ion beam sputtering of organic mixtures for peptides and oligonucleotides up to masses on the order of 10 000 Da. This matrix-enhanced SIMS exhibits substantial increases in the ionization efficiency of selected analyte molecules compared to conventional SSIMS processes. Thus, higher mass peptides, proteins, and nucleic acids become accessible to near-surface analysis by ion beam techniques, and subpicomole sensitivity has been demonstrated. A number of matrices were examined for their efficiency in ME-SIMS applications, and these initial matrix studies focused on common MALDI matrices and their isomers. The results of this survey indicate that 2,5-dihydroxybenzoic acid provides the best general enhancement of molecular secondary ions emitted from analyte/matrix mixtures. One of the most fundamental measurements necessary for elucidation of the structure of biomolecules is the determination of molecular weight, and mass spectrometry has long been one of the best techniques for directly obtaining this information. Conventional mass spectrometry using electron impact and chemical ionization requires vaporization of neutral moieties followed by ionization. The vaporization process inherently limits the applicability of these techniques to analyses of small compounds, thus excluding high-mass biomolecules, which are typically large, nonvolatile, and/or thermally labile. In the past two decades, conceptually new techniques have been developed to overcome this limitation. Examples of these new techniques include field desorption mass spectrometry (FDMS),1 secondary ion mass spectrometry (SIMS),2 fast atom bombardment mass spectrometry (FAB-MS),3 plasma desorption mass spectrometry (PDMS),4 electrospray ionization mass spectrometry (ESI-MS),5 and matrix-assisted laser desorption/ionization mass spectrometry (1) Winkler, H. U.; Beckey, H. D. Biochem. Biophys. Res. Commun. 1970, 46, 391. (2) Cooks, R. G.; Amy, J.; Bier, M.; Schwarz, J.; Schey, K. In Advances in Mass Spectrometry; Longevaille, P., Ed.; Heyden & Son: London 1989; Vol. 11A. (3) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N. J. Chem. Soc., Chem. Commun. 1981, 325. (4) Torgerson, D. F.; Skowronski, R. P.; Macfarlane, R. D. Biochem. Biophys. Res. Commun. 1974, 60, 616. 0003-2700/96/0368-0873$12.00/0
© 1996 American Chemical Society
(MALDI-MS).6,7 Among these developments, the most important breakthroughs for organic and biological applications have occurred with advances in the ESI and MALDI techniques. One of the most important factors for continued technical progress in the mass spectrometry of biomolecules is innovative developments in desorption and ionization.8 Although applications of MALDI have grown dramatically over the past few years, the fundamental mechanisms of ionization in MALDI are still not well understood. Even with this limited understanding, methods for improving the performance of MALDI have led to the discovery of new matrices which enhance the ionization of selected classes of biopolymers.9-11 SIMS techniques have been extensively applied to both elemental and molecular analyses of nonvolatile materials. SIMS is normally divided into two distinct classes, known as dynamic and static SIMS.12 Dynamic SIMS employs relatively intense primary ion beams which erode the sample surface at sputtering rates ranging between 0.1 and 10 nm/s. These high sputter rates produce predominately elemental ions or low-mass cluster ions. Combining high sputter rates with ion yield enhancements produced by reactive primary ions such as O2+ (for positive ion analysis) and Cs+ (negative ion analysis), dynamic SIMS achieves parts-per-million (ppm) to parts-per-billion (ppb) detection sensitivities for most elements. As a consequence of its high detection sensitivity, dynamic SIMS has been successfully applied to elemental analysis in material science,13 as well as to trace element analysis in geological and biological sciences.14,15 The other form of SIMS, known as static SIMS (SSIMS), employs very low intensity primary ion beams to sputter the top monolayer of a surface.16 Secondary ions produced under static SIMS conditions are typically elemental, molecular, and molecular fragments, (5) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. K.; Whitehouse, C. Science 1989, 246, 64. (6) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1988, 78, 53. (7) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151. (8) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1994, 66, 634R. (9) Beavis, R. C. Org. Mass Spectrom. 1992, 27, 653. (10) Fitzgerald, M. C.; Parr, G. R.; Smith, L. M. Anal. Chem. 1993, 65, 3204. (11) Wu, K. J.; Stedding, A.; Becker, C. H. Rapid Commun. Mass Spectrom. 1993, 7, 142. (12) Benninghoven, A.; Rudenaur, F. G.; Werner, H. W. Secondary Ion Mass Spectrometry: Basic Concepts, Instrumental Aspects, Applications and Trends; John Wiley and Sons: New York, 1987. (13) Wilson, R. G.; Stevie, F. A.; Magee, C. W. Secondary Ion Mass Spectrometry: A Practical Handbook for Depth Profiling and Bulk Impurity Analysis; Wiley Interscience: New York, 1989. (14) Russell, W. A.; Papanastassiou, D. A.; Trombello, D. A. Geochim. Cosmochim. Acta 1978, 42, 1075. (15) Represetative articles: Biol. Cell 1992, 74.
Analytical Chemistry, Vol. 68, No. 5, March 1, 1996 873
characteristic of the near-surface composition. Static SIMS is generally performed using either quadrupole mass filters or timeof-flight (TOF) mass analyzers, and the advent of high-performance TOF-SIMS instrumentation has provided dramatically improved capabilities in near-surface chemical analysis by static SIMS. For example, commercial TOF-SIMS instrumentation normally operates at primary ion doses ranging between 1010 and 1012 ions/cm2, corresponding to removal of 10-4-10-2 monolayers assuming a sputter yield of 10 and a molecular surface density of 1015 molecules/cm2. The ion optics in these systems achieve high ion transmission efficiencies at mass resolutions (M/∆M, full width at half-maximum, fwhm) in excess of 10 000 using either high-performance ion reflectors17 or triple electrostatic energy analyzers.18 Molecular and elemental detection sensitivities as low as ppm levels are achieved within the top monolayers of a surface with this technique. Plasma desorption mass spectrometry uses fast heavy ions (MeV) from fission fragments to sputter secondary ions electronically from samples of neat biomolecules.4 Proteins with masses up to a few tens of kilodaltons have been detected by PDMS.19 In particular, the nitrocellulose membrane has been found to be very helpful in enhancing molecular ion formation.20 Extensive studies have also been carried out in an effort to understand of the dynamics of PDMS ion sputtering and ejection processes.21,22 Another mass spectrometric technique that provides molecular analysis of large, thermal labile biomolecules is fast atom bombardment (FAB), which employs an energetic neutral beam to sputter the surface of a liquid matrix, into which the analyte molecules are dissolved or dispersed.3 If an energetic ion beam is used instead of a fast neutral beam to sputter the liquid surface (a technique referred to as liquid SIMS or LSIMS), this configuration is essentially SIMS of a liquid surface. An obvious question that arises in considering the experimental configurations of the MALDI, SIMS, and FAB techniques is whether biomolecular ions can be produced by ion beam sputtering of a solid mixture composed of a matrix and an analyte. Busch and co-workers demonstrated over 10 years ago that such matrix-assisted SIMS produced relatively intense signals from a solid mixture of low-mass quaternary ammonium salts mixed with an ammonium chloride matrix.23 Gillen et al. also demonstrated enhanced secondary ion emission of small biomolecules from a frozen glycerol matrix.24 However, there has not been any report demonstrating formation and detection of high-mass (>several kilodaltons) biomolecular ions produced by keV ion beam sputtering a solid sample surface.
This latter observation leads to the obvious question concerning the possible existence of mechanistic differences between sputtering of liquids (FAB, LSIMS) and solids (SIMS). All three techniques typically use primary particles having keV energies. Secondary ion ejection produced by keV particle bombardment is sometimes referred to as nuclear sputtering, and the ejection of particles in these processes has been modeled using a pressurepulse mechanism in which a strong energy density gradient is rapidly established in space and time.25 Thus, neutral sputtering mechanisms from liquid and solid surfaces should be similar. However, the ionization processes occurring in solid SIMS and FAB (LSIMS) are probably quite different because of differences in matrix effects between liquid and solid mixtures, as well as the higher total sputter yields produced by ion bombardment of liquid surfaces compared to the yields produced from bombardment of a solid. In addition, FAB and LSIMS generally produce ions at significantly higher masses than those observed in solid SIMS of pure compound residues, because the liquid matrix rapidly dissipates most of the incident particle energy and thus minimizes primary ion (or neutral) fragmentation of the analyte. The results presented in this paper demonstrate that highmass molecular ions can be produced by energetic ion beam sputtering of solid mixtures of analytes and suitable matrices, which is essentially static TOF-SIMS analysis of samples prepared by MALDI preparation methods. It combines the intrinsic surface sensitivity of static SIMS with a FAB-like matrix enhancement concept and MALDI’s well-developed matrix/analyte sample preparation protocols. This new approach, which we refer to as matrix-enhanced secondary ion mass spectrometry (ME-SIMS), takes advantage of the attributes of the SIMS, FAB, and MALDI techniques. The basis of this innovative technique for generating mass spectra of analyte molecules lies in the matrix-enhanced desorption/ionization process. In this work, we present applications of ME-SIMS in analyses of peptides, proteins, and oligonucleotides. This research demonstrates that molecular ions of high-mass biomolecules that cannot be formed or detected by standard static SIMS techniques are readily formed by this new process. The most significant improvement of ME-SIMS is its ability to expand the mass range of static SIMS techniques out to masses greater than 10 000 Da. One of the key issues associated with ME-SIMS is the selection of appropriate matrix material for different analytes. We have studied several compounds for their effectiveness as a matrix as well as the general characteristics of the ME-SIMS technique. The initial survey shows that 2,5-dihydroxybenzoic acid is the most efficient matrix found to date for ME-SIMS.
(16) Vickerman, J. C.; Brown, A.; Reed, N. M. Secondary Ion Mass Spectrometry: Principles and Applications; Clarendon Press: Oxford, 1989. (17) Niehuis, E. In Secondary Ion Mass SpectrometrysSIMS VIII; Benninghoven, A., Janssen, K. T. F., Tumpner, J., Werner, H. W., Eds.; John Wiley & Sons: New York, 1992. (18) Schueler, B. Microsc. Microanal. Microstruct. 1992, 3, 1. (19) Jonsson, G.; Hedin. A.; Håkansson, P.; Sundqvist, B. U. R.; Vennich, H. and Roepstorff, P. Rapid Commun. Mass Spectrom. 1989, 3, 190. (20) Jonsson, G.; Hedin. A.; Håkansson, P.; Sundqvist, B. U. R.; Nielsen, F.; Roepstorff, P.; Johansson, K.; Kamensky, I.; Lindberg, M. Anal. Chem. 1986, 58, 1084. (21) Fenyo, D.; Sundqvist, B. U. R.; Karlsson, B.; Johnson. R. E. Phys. Rev. B. 1990, 42, 1895. (22) Sundqvist, B. U. R. Anal. Chim. Acta 1991, 247, 265. (23) Busch, K. L.; Hsu, B. H.; Xie, Y. X.; Cooks, R. G. Anal. Chem. 1983, 55, 1157. (24) Gillen, G.; Christiansen, J. W.; Tsong, I. S. T.; Kumball, B.; Williams, P. Rapid Commun. Mass Spectrom. 1988, 2, 67.
EXPERIMENTAL SECTION Materials and Reagents. Peptide and protein samples were purchased from Sigma (St. Louis, MO) with purity greater than 90%. They were analyzed as received without further purification. All the matrices were obtained from Aldrich (Milwaukee, WI). Single-stranded mixed-base oligonucleotides were custom synthesized and purified (HPLC) by Keystone Laboratories (Menlo Park, CA). Sample Preparation. Peptides, proteins, and nucleic acids were used as test molecules to study the matrix-enhanced ionization process. Sample preparations were directly adopted
874
Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
(25) Johnson, R. E.; Sundqvist, B. U. R. Rapid Commun. Mass Spectrom. 1991, 5, 574.
from existing MALDI sample preparation protocols. All the matrices used in this report were dissolved in 50% H2O and 50% acetonitrile to a concentration of ∼0.5 M, and matrix solutions were prepared fresh daily. Analytes were dissolved in deionized water to a concentration of ∼10-4 M. Sample solutions consisted of equal volume mixtures (5 µL each) of the matrix and analyte solutions. Some mixture solutions were treated with cation exchange resins (BioRad 50W-X8, mesh size 100-200 µm) for a few minutes to reduce Na adduct formation. A 0.2 µL aliquot of the matrix/analyte mixture was pipetted onto a sample substrate. The typical amount of analyte in each sample was about 10 pmol, an amount comparable to that used for standard MALDI analysis. A clean 1 cm × 1cm Si wafer was chosen as the sample substrate because of its flatness. To avoid cross contamination between samples, fresh substrates were used for each analysis. Droplets typically spread out on the surface to an area of ∼2 mm in diameter, with drying taking place in air at room temperature prior to insertion into the vacuum system. Dried samples typically contained large crystals, and the most intense analyte ion signals are usually obtained from the surfaces of these bulky crystals. Additional sample preparation procedures have also been explored, including spin-coating thin sample films onto a Si wafer. The objective of this work was to produce very thin (several monolayers thick) coatings of matrix/analyte mixtures. By adjusting the rotating speed between 1000 and 3000 rpm and increasing the acetontrile concentrations, we were able to prepare thin samples over an area of ∼1 cm2. These relatively uniform samples were composed of a flat film in the center portion of the sample and thick crystals on the edge of the dried compound. The most intense signals for both matrix and analyte ions were usually obtained from these thick crystals. Since equivalent signals could be produced from crystals prepared using normal MALDI procedures, this thin film preparation approach was abandoned. The results presented in this paper were obtained from solid residues prepared by standard MALDI sample preparation techniques. TOF-SIMS Instrument. The mass spectrometer used was a Charles Evans & Associates time-of-flight secondary ion mass spectrometer (TRIFT). A detailed description of the apparatus and its principles of operation have been published elsewhere,18 and only a brief overview of the experimental setup is given herein. The instrument was designed for elemental and molecular ion analysis at high mass resolution, mass measurement accuracies, and detection sensitivities. The TRIFT system incorporates a pulsed Cs+ primary ion source, and the nominal impact energy of the beam on the sample surfaces was 11 keV. Typical pulse repetition rates for these analyses were 5 kHz, with a subnanosecond primary ion pulse width. This pulse repetition rate permitted analysis out to masses >20 000 Da, and the subnanosecond pulse widths assured mass resolutions (M/∆M) on the order of ∼5000 at mass 41 Da. The dc primary ion beam intensity at the sample was a few nanoamperes, and the operating spot size was ∼50 µm in diameter. No charge neutralization was used, since there was no significant sample charging during the MESIMS measurements. The base pressure of the chamber was 3 × 10-9 Torr, and the normal working pressure was 1 × 10-8 Torr. Secondary ions generated on the surface were extracted using a 1.12 V/µm extraction field and accelerated to an energy of 4.5 keV. These ions were focused and transported by two sets of ion optical lenses into the electrostatic sectors. The TOF mass
analyzer has an effective flight length of 2 m, which consists of three 90° quasi-hemispherical electrostatic sector analyzers (ESAs) in the ion flight path and a dual microchannel plate for ion detection. Specialized stigmatic ion optics extract and transport ions from the sample surface to the TOF mass analyzer, and complete first-order energy focusing of ion flight times is accomplished by the ESAs. The spectrometer corrects and compensates flight time variations introduced by kinetic energy aberrations in the acceleration region as well as flight time differences arising from angular variances. Secondary ions were postaccelerated by 10 kV to increase the detection sensitivity for high-mass particles; the secondary ions’ impact energy on the detector was ∼14.5 keV. Spectra were acquired in single-ion counting mode using a custom-built multistop time-to-digital converter having 138 ps timing resolution. Typical data acquisition times averaged between 30 s and 5 min. Positive ion mass spectra only are reported in this paper. RESULTS AND DISCUSSION Figure 1a shows a positive ion TOF-SIMS mass spectrum of porcine renin substrate (average MW ) 1759) with 2,5-dihydroxybenzoic acid (2,5-DHB) as a matrix material. The matrix-to-analyte ratio in the mixture solution was ∼10 000:1, with 1 pmol of analyte loaded for analysis. The total acquisition time was 2 min. The detected peaks correspond to (M + H)+ of analyte ions, and the renin substrate molecular ion signal produced in ME-SIMS was very stable over a wide area of the sample. This characteristic is quite different from the ionization produced by MALDI, which normally requires scanning for good spots in order to obtain strong analyte signals. As a consequence of this more spatially uniform ionization, matrix-enhanced SIMS often produces more stable signals, giving rise to better counting statistics than observed in similar analyses performed by MALDI. The isotopic features are clearly resolved, and mass resolution was measured to be M/∆M ) 6000 (fwhm). This measured isotope distribution is in good agreement with the calculated pattern shown in the inset in Figure 1a. TOF-SIMS analyses were also performed on a neat resin substrate sample deposited directly onto a Si wafer. The mass spectrum obtained from this neat residue is shown in Figure 1b. The total secondary ion signal intensity produced from this neat residue is a factor of 50 lower than the intensity measured in the ME-SIMS spectrum. Therefore, the ionization yield of this analyte is greatly enhanced by the presence of the matrix, although the matrix molecules are no longer used for the absorption of laser energy as in MALDI. In addition to the low anayte ion yield from the neat residue, more significant fragmentation of the analyte is observed in the spectrum of the neat residue than is observed from the matrix-surrounded sample. The lower fragmentation of the analyte in ME-SIMS is attributed to the nestle environment of the solid matrix providing a much softer yet efficient desorption and ionization of this compound. As is discussed in more detail below, this enhancement in secondary ion formation is strongly dependent upon the matrix employed, the matrix/analyte concentration ratio, the sample mixture crystal formation, and the primary ion beam dose. The initial selection of matrix materials focused on existing MALDI matrices such as 2,5-DHB, 4-hydroxy-R-cyanocinnamic acid (4-HCCA), sinapanic acid, and 3-hydroxypicolinic acid (3HPA). These materials were chosen because MALDI sample preparation protocols are well developed and documented, and Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
875
Figure 1. (a) Positive ion TOF ME-SIMS mass spectrum of porcine renin substrate (calculated MW ) 1759) with 2,5-dihydroxybenzoic acid as a matrix material. Total analyte loaded for analysis was 1 pmol, and data acquisition time was 2 min. M/∆M (fwhm) ) 6000. The inset shows the calculated isotope distribution. (b) Positive ion TOF-SIMS mass spectrum obtained from neat porcine renin substrate on Si. Data acquisition time was 2 min, and 1 pmol of analyte was loaded for analysis.
MALDI matrices are known to be good ionization agents for biomolecules. In MALDI applications, matrix molecules are believed to perform several important functions, including absorption of the photon energy, initiation of the desorption event(s), and ionization of neutral analytes. In the ME-SIMS process, by contrast, there are no photochemical reactions initiated by irradiating the sample with UV laser photons. The matrix molecules, however, do play a significant role in promoting the ejection and ionization of the analytes. Therefore, the presence of strong proton donors, such as those used as MALDI matrices, should efficiently promote the cationization of neutral molecules sputtered from a solid mixture by an energetic ion beam. A positive ion ME-SIMS mass spectrum of human β-endorphin (MW ) 3465) is shown in Figure 2a. The matrix was 2,5-DHB, and 5 pmol of analyte was used for analysis. The inset shows a resolved isotope distribution of the molecular ion peaks, and the corresponding mass resolution is M/∆M ) 8000. A small amount of fragmentation is observed at [M - 17]+. Intact molecular ions of β-endorphin are not produced from neat residues using static SIMS conditions, as is demonstrated in Figure 2b. Figure 3a is a positive ion mass spectrum produced in the matrix-enhanced process from a bovine insulin (MW ) 5733.5) 876 Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
residue mixed with 2,5-DHB. The total data acquisition time was 30 s. Singly and doubly charged (protonated) insulin ion peaks are detected. The isotopic distribution is partially resolved at this mass, indicating a mass resolution of M/∆M ≈ 5000. Similar to the result from β-endorphin, a daughter ion at [M - 17]+ is also observed. This feature has been reported in MALDI analysis of bovine insulin, and the [M - 17]+ was attributed to metastable decay in the field-free drift region.26 Recent MALDI studies in our laboratory also indicate that the degree of such fragmentation is strongly dependent upon the matrix used.27 For example, a much lower intensity [M - 17]+ peak was produced by MALDI analysis of bovine insulin using 3-HPA as the matrix. Small amounts of matrix adducts [M + DHB]+ were also detected in the ME-SIMS spectrum. Bovine insulin does not form molecular ions in the absence of a matrix, as indicated in Figure 3b, which is a conventional static SIMS spectrum of a neat residue of bovine insulin on a Si substrate. Attempts to generate molecular ion signals by conventional static SIMS techniques using an etched (26) Zhou, J.; Ens, W.; Standing, K. G.; Verentchikov, A. Rapid Commun. Mass Spectrom. 1992, 6, 71. (27) Wu, K. J.; Odom, R. W., to be published.
Figure 2. (a) Positive ion TOF ME-SIMS mass spectrum of human β-endorphin (calculated MW ) 3465) with 2,5-dihydroxybenzoic acid as a matrix material. Total analyte loaded for analysis was 5 pmol, and data acquisition time was 1 min. M/∆M (fwhm) ≈ 8000. (b) Positive ion TOF-SIMS mass spectrum of neat human β-endorphin on Si substrate. Data acquisition time was 2 min, and 1 pmol of analyte was loaded for analysis. No analyte ion signal was observed.
Ag foil as a substrate produced results similar to those shown in Figure 3b. Conventional static SIMS is capable of producing protonated ions from neat peptides on Si substrates up to peptide masses of ∼2500 Da, and a slightly higher mass range can be achieved using etched Ag foil as the substrate. For all analyte systems investigated to date, ME-SIMS provides greater ionization and reduced fragmentation in biomolecule analysis than is possible using conventional static SIMS, irrespective of the preparation and composition of the static SIMS substrate. Figure 4 shows a positive ion mass spectrum of bovine ubiquitin (MW ) 8565). Isotope peaks are no longer resolved, and the mass resolution is primarily limited by the width of the isotopic envelope. The total amount of ubiquitin deposited for analysis was 5 pmol, and the matrix used was 2,5-DHB. It is found that the rate of secondary analyte ion formation by ME-SIMS decreases as the mass of the analyte increases; therefore, higher mass molecules generally require longer acquisition times. This mass-dependent secondary ionization rate is attributed to changes in both the sputter and ionization yields as a function of analyte mass.
The largest molecule we have successfully analyzed to date was chicken egg lysozyme, having a molecular mass of >14 000 Da. The molecular ion signals were much more difficult to detect, and the spectrum showed a weak and broad mass distribution for lysozyme ions instead of the distinct peak seen in previous figures. Mass resolution in this measurement was ∼200, which is significantly lower than the resolution obtained from lower mass samples. The reasons for this significant degradation in mass resolution include possible sample contamination from impurities resulting in excessive adduct formation, extensive decomposition during sputtering, and low sputter/ionization yields. Improvements could be made by applying different sample preparations and using a more effective matrix. We are currently exploring these two possibilities in detail. If the poor resolution in the spectrum is caused by ion beam damage, metastable formation, or low sputter yields, the configuration of the sputtering process (primary species, impact energy) will have to be optimized in order to improve the spectrum. As exemplified in the analysis of lysozyme, the higher the mass of the analyte, the more difficult it is to produce intact molecular ions by direct ion beam bombardment of the solid surface. Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
877
Figure 3. (a) Positive ion TOF ME-SIMS mass spectrum of bovine insulin (calculated MW ) 5733) with 2,5-dihydroxybenzoic acid as a matrix material. Total analyte loaded for analysis was 10 pmol, and data acquisition time was 30 s. (b) Positive ion TOF-SIMS mass spectrum of neat bovine insulin on Si substrate. Data acquisition time was 2 min, and 10 pmol of analyte was loaded for analysis. No analyte ion signal was observed.
Single-stranded oligonucleotides have always been more difficult to analyze by MALDI than peptides and proteins due to low ionization efficiency, severe base losses, and phosphodiester backbone fragmentation. To examine the applicability of MESIMS for nucleic acid analysis, we have investigated a number of mixed-base oligonucleotides in the size range between 4-mer and 35-mer. An example of the positive ion mass spectrum of an underivatized, custom-synthesized mixed-base single-stranded DNA 26-mer (MW ) 8157) is illustrated in Figure 5. The matrix was 2,5-DHB, 15 pmol of analyte was loaded, and the spectrum was accumulated in 2.5 min. Isotopic features were not resolved, and the mass resolution is limited by the isotope envelope to M/∆M ≈1500. For comparison, ME-SIMS analyses were also performed on this 26-mer using 3-hydroxypicolinic acid as the matrix. The molecular ion signal from this 26-mer in 3-HPA was much weaker than the signal produced using 2,5-DHB as the matrix. Our results from analysis of several other oligonucleotides consistently demonstrated that 2,5-DHB is a much more effective matrix than 3-HPA in ME-SIMS. Typically, we observe a factor of 10 or more increase in molecular ion signals from oligonucleotides on going from a 3-HPA to a 2,5-DHB matrix. 878
Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
We also performed MALDI measurements on these two 26mer samples after the ME-SIMS experiments. MALDI produced the opposite results, i.e., 2,5-DHB gave almost no 26-mer molecular ion signal by MALDI, whereas a very intense 26-mer peak was produced with 3-HPA as the matrix. These results demonstrate that ME-SIMS and MALDI do not always share the same matrices. Since the fundamental ionization/ejection mechanisms are quite different for these two processes, it is not surprising that these techniques have different optimum matrices. The drawback of using 2,5-DHB as a matrix for nucleic acid analysis is the lower pH (1.9) of its solutions, which usually causes severe multiple base losses, as illustrated in Figure 5. Some of the base loss can be produced by the cation exchange resins added in the matrix/ analyte mixutre solution to remove the alkali metal adducts. These results from ME-SIMS measurements also suggest base losses as often observed by MALDI are caused by high acidic environment rather than photodissociation induced by the laser. A series of comparative measurements have been carried out to study matrix effects in ME-SIMS. We examined the efficiency of different matrices for generating molecular ions of various analytes. Sample analytes included substance P (MW ) 1347), β-endorphin (MW ) 3465), human growth hormone releasing
Figure 4. Positive ion TOF ME-SIMS mass spectrum of bovine ubiquitin (calculated MW ) 8565) with 2,5-dihydroxybenzoic acid as a matrix material. Total analyte loaded for analysis was 5 pmol, and data acquisition time was 3 min. Isotope peaks are not resolved, and mass resolution is limited by the width of the isotopic envelope, M/∆M ) 1500.
Figure 5. Positive ion TOF ME-SIMS mass spectrum of single-stranded oligonucleotide 26-mer. Sequence: 5′-GAGCTCGAAGTGGCACTAGTGGGTAA (MW ) 8157). Total analyte loaded for analysis was 15 pmol, and data acquisition time was 2.5 min.
factor (MW ) 5040), bovine insulin (MW ) 5733), equine cytochrome c (MW ) 12 360), DNA 4-mer (MW ) 1156), and a DNA 18-mer (MW ) 5546). Three classes of matrices were tested for their effectiveness in generating molecular ions from these compounds: 4-hydroxy-R-cyanocinnamic acid; 2,5-dihydroxy-
benzoic acid and its isomers 2,4-DHB, 2,6-DHB, and 3,5-DHB; and 3-hydroxypicolinic acid and its isomers 2-hydroxypyridine-5carboxylic acid (HPCA) and 2-hydroxynicotinic acid (HNA). To ensure proper comparison, all matrix/analyte mixtures were prepared at concentration ratios of ∼10 000:1, and 20 pmol of Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
879
Table 1. Ion Yields from Different Matrices Using MALDI and ME-SIMS Techniquesa matrix analyte substance P β-endorphin GRFg insulin cytochrome c 4-mer 18-mer
technique
neat
4-HCCAb
MALDI ME-SIMS MALDI ME-SIMS MALDI ME-SIMS MALDI ME-SIMS MALDI ME-SIMS MALDI ME-SIMS MALDI ME-SIMS
W W -
S S S W S W S W S -
2,5-DHBc
3-HPAd
2,4-DHBe
HNAf
S S S S S S S S S W W S S
S S S M S W S M S S W S -
S S M M M W M W W W
M M M M M -
a S, signal > 10 000 counts; M, 1000 < signal < 5000 counts; W, signal < 100 counts; -, no signal (for ME-SIMS only). b 4-Hydroxy-R-cyanocinnamic acid. c 2,5-Dihydroxybenzoic acid. d 3-Hydroxypicolinic acid. e 2,4-Dihydroxybenzoic acid. f 2-Hydroxynicotinic acid. g Human growth hormone releasing factor.
analyte was loaded for analysis. These freshly prepared samples were first analyzed by ME-SIMS, after which MALDI analyses were performed on the same instrument. Table 1 summarizes the ionization yields for some of the above-mentioned matrices. The results of this study clearly show that 2,5-DHB is the most efficient matrix for ME-SIMS of the peptides and oligonucleotides studied. This preliminary survey also suggests that analyte ionization by ion beam sputtering depends more critically on matrix composition than it does in MALDI analysis. This higher degree of matrix dependence is undoubtedly due to the requirements not only that the matrix provide a nestle environment about the analyte molecules and a source of protons for ion formation (both of which are required in MALDI) but also that the matrix/ analyte mixture have a sufficiently high concentration of analytes in the top monolayer(s) of the surface. Thus, the ideal matrix for ME-SIMS is one that can readily donate protons to the analyte (for positive ion formation) and one that forms a solid solution whose surface concentration is enriched with analyte. Since 2,5-DHB is a very effective MALDI matrix for peptides and proteins, it is not too surprising that 2,5-DHB works well in ME-SIMS for these analytes. The most significant discrepancy between MALDI and ME-SIMS occurs in DNA analyses. The most effective MALDI matrix for oligonucleotides (3-HPA) is clearly not the optimum matrix for ME-SIMS, but rather 2,5-DHB produces the highest ionization enhancement of oligonucleotides using energetic ion beam sputtering. Therefore, the optimum matrices for ME-SIMS and MALDI analyses of a given analyte may be different. Since these two techniques have different ejection and presumably different ionization mechanisms, there is no reason to expect, a priori, that these very different processes should be optimized within common matrices. Furthermore, the difference in matrix selection for oligonucleotides implies that 2,5-DHB is a good ionization agent for DNA in aqueous solutions (and perhaps also in solid mixtures), as demonstrated by the ME-SIMS results. Therefore, unsuccessful results from 2,5-DHB isomers primarily arise from the variation in chemical properties among each of the isomers. Such differences are enough to hinder ionization of the nucleic acids. We have also studied the effects of varying the pH of different matrices on the ionization by ME-SIMS. The studies involved adjusting the pH of matrix solutions by adding HCl, and two sets 880 Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
of pH values (pH ) 1.0 and 2.0) were investigated in detail with regard to the ion yields of various peptides and oligonucleitides mixed with these matrices. The analyte molecular ion signals for the peptide samples were comparable at these two pH values; thus, lowering the pH does not significantly change the degree of ionization of these analytes. The nucleic acid samples, however, exhibit extensive base loss in the lower pH matrix, indicating that these oligonucleotides are much more sensitive to acidic environments. By contrast, increasing the pH does substantially degrades analyte peak intensities for both peptides and oligonucleotides. A total loss in analyte signal is normally observed with neutral (pH ) 7) matrix solutions for both types of biomolecules. The effects of different matrix-to-analyte concentration ratios on the ME-SIMS results were also examined. We prepared matrix/analyte mixtures ranging from a unit molar ratio mixture (M:A ) 1:1) to extremely dilute analyte solutions (M:A ) 109:1). The results indicate that ME-SIMS operates over matrix/analyte ratios between 103:1 and 106:1. At ratios higher than 106:1, the analyte surface concentration approaches the limit of static SIMS detection sensitivity, whereas for ratios lower than 103:1, the analyte surface concentration becomes too high for ME-SIMS to work effectively. Detection sensitivity depends, in general, on the type of analyte and the molecular mass of the sample. Higher detection sensitivity was obtained for proteins and peptides than for nucleic acids. A decreasing trend in sensitivity as a function of analyte mass is also observed. The smallest amount of analyte loaded and successfully analyzed by ME-SIMS technique was 50 fmol of bovine insulin using 2,5-DHB as a matrix material. In addition to matrix effects, matrix/analyte concentration ratios, and sample preparation techniques, the secondary ion signal intensity in ME-SIMS is also a function of the primary ion beam dose. Ion yields as a function of primary ion dose were evaluated for several analytes having masses between 2000 and 10 000 Da. The matrix used was 2,5-DHB, and the matrix/analyte molar ratio was 105:1. A pulsed primary beam with a 50 µm diameter spot size was used, and the secondary ion signal intensity was evaluated by integrating the intensity of the singly charged molecular ion peak as a function of analysis time, in which each integration period was 20 s. An example of this measurement using bovine insulin as analyte is shown in Figure 6. A decay time constant of ∼60 s was found on the (M+H)+ ion signal. This
Figure 6. Measurements of bovine insulin molecular ion intensity decay as a function of time exposed to Cs+ ion beam on a fixed spot. The signal intensity is calculated by integrating the intensity of the singly charged molecular ion peak as a function of analysis time. Each integration period was 20 s.
decay time does not depend on analyte mass for constant operating conditions of the primary ion beam. This observation indicates that the primary beam, as operated under typical conditions for ME-SIMS, requires about 60 s to damage the top matrix/analyte mixture surface and reach the static SIMS limit on a fixed spot. Simply moving the ion beam to a fresh spot regains the sample signal. Therefore, analytically, the high primary ion doses do not pose significant limitations to the technique. Within the range of matrices evaluated to date, the upper mass limit achieved by ME-SIMS is >10 000 Da. Increasing the detectable mass range will require finding more effective matrices and developing new sample preparation and (possibly) analysis techniques. The matrix-enhanced process has also been applied to the analysis of organic polymers such as poly(ethylene glycols) and polycarbonates, and preliminary results for oligomers of these compounds indicate that ME-SIMS provides higher detectable mass ranges and better detection sensitivity than conventional static SIMS. In addition, the matrix-enhanced process may provide more accurate information on molecular weight distributions of low-mass synthetic oligomers than can be currently obtained by SSIMS techniques. From a macroscopic point of view, the difference between MALDI and ME-SIMS lies in the primary source of excitation used for the generation of the secondary ions. MALDI uses a pulsed laser, and ME-SIMS employs a beam of energetic ions. Various investigations have been performed to study the fundamentals of both desorption and ionization in MALDI processes,28-30 and it is generally accepted that analyte ions generated in MALDI are produced by gas phase ionization mechanisms. A plume of ions and neutrals is formed by laser irradiation, and collisions within the expansion plume create a supersonic jet of ions and neutrals, which is demonstrated by the essentially mass independent initial (28) Wang, B. H.; Dreisewerd, K.; Bahr, U.; Karas M.; Hillenkamp, F. J. Am. Soc. Mass Spectrom. 1993, 4, 393. (29) Kinsel, G. R.; Gilling, K.; Edmondson, R.; Russel, D. H. Proceedings of the 42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 29-June 3, 1994; p 4. (30) Bo¨kelmann, V.; Spengler, B; Kaufmann, R. Eur. Mass Spectrom. 1995, 1, 81.
velocities of MALDI ions.31-34 Collisions between neutral analytes and matrix ions are thought to produce a significant fraction of analyte ions. Such a model suggests that most analytes are ejected as neutrals and are subsequently ionized above the sample surface. A very different process must occur in matrix-enhanced SIMS, since ∼105 primary ions strike the surface in each primary pulse, generating between 10 and 100 secondary ions under typical SIMS conditions. Therefore, there are simply not enough secondary ions or sputtered neutrals to produce significant collision-induced gas phase ionization of neutral analytes. The analyte ions detected by ME-SIMS may exist as preformed ions; i.e., protonated species present in the solid mixture prior to the sputter ejection from the surface or protonated analyte ions could be formed in the very near-surface (the so-called self-edge) region as the sputtered species is released from the matrix lattice. The data presented in this paper have not addressed these mechanistic issues, and further studies on the dynamics and energetics of secondary ion ejection from solid mixtures are required to improve the understanding of the matrix-enhanced ionization process. This improved understanding could also provide valuable insight into MALDI ionization mechanisms. CONCLUSIONS The results presented in this paper demonstrate that highmass molecular ions can be produced by energetic ion beam sputtering of solid mixtures of analytes contained within or surrounded by suitable matrices. This ME-SIMS technique combines the intrinsic surface sensitivity of static SIMS with a FAB-like matrix enhancement concept and MALDI’s well developed matrix/analyte sample preparation protocols. Biopolymers such as peptides, proteins, and oligonucleotides have been successfully analyzed with this technique at a subpicomole detection sensitivity. A number of compounds have also been examined for their effectiveness as matrices for ME-SIMS. This (31) Beavis, R. C.; Chait, B. T. Chem. Phys. Lett. 1991, 181, 479. (32) Pan, Y.; Cotter, R. J. Org. Mass Spectrom. 1992, 27, 3. (33) Huth-Fahre, T.; Becker, C. H. Rapid Commun. Mass Spectrom. 1991, 5, 378. (34) Ens, W.; Mao, Y.; Mayer, F.; Standing, K. G. Rapid Commun. Mass Spectrom. 1991, 5, 117.
Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
881
initial survey indicates that 2,5-dihydroxybenzoic acid is the most efficient matrix. This matrix study also suggests that ME-SIMS shares common matrices with MALDI, but discrepancies do exist. 3-HPA does not produce strong nucleic acid ion signals in MESIMS, but 2,5-DHB generates the most intense secondary analyte ions from oligonucleotides by primary ion beam bombardment. The matrix-enhanced process described in this paper extends the surface analysis capabilities of conventional static SIMS to the analysis of higher molecular weight biomolecules and organic polymers. Molecular ion masses >10 000 Da have been detected by this technique, and further research is required to determine the upper mass limit of this matrix-enhanced ionization process. Thus, ME-SIMS enables static SIMS techniques to analyze a
882
Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
broader range of organic solids, including biomolecules and synthetic polymers, having higher molecular weights than can currently be analyzed by ion beam sputtering techniques. ACKNOWLEDGMENT Financial support in part by the National Science Foundation (NSF), Small Business Innovation Research (SBIR) Grant ISI9160545, is greatly appreciated. Received for review July 18, 1995. Accepted November 30, 1995.X AC950717I X
Abstract published in Advance ACS Abstracts, January 15, 1996.
