Ionization Mass Spectrometry

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ac research

Anal. Chem. 2010, 82, 11–15

Letters to Analytical Chemistry Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Method for Selectively Producing Either Singly or Multiply Charged Molecular Ions Sarah Trimpin,*,† Ellen D. Inutan,† Thushani N. Herath,† and Charles N. McEwen‡ Wayne State University, Department of Chemistry, Detroit, Michigan 48202, and The University of the Sciences in Philadelphia, Department of Chemistry and Biochemistry, Philadelphia, Pennsylvania 19104 Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is noted for its ability to produce primarily singly charged ions. This is an attribute when using direct ionization for complex mixtures such as protein digests or synthetic polymers. However, the ability to produce multiply charged ions, as with electrospray ionization (ESI), has advantages such as extending the mass range on mass spectrometers with limited mass-tocharge (m/z) range and enhancing fragmentation for structural characterization. We designed and fabricated a novel field free transmission geometry atmopsheric pressure (AP) MALDI source mounted to a high-mass resolution Orbitrap Exactive mass spectrometer. We report the ability to produce at will either singly charged ions or highly charged ions using a MALDI process by simply changing the matrix or the matrix preparation conditions. Mass spectra with multiply charged ions very similar to those obtained with ESI of proteins such as cytochrome c and ubiquitin are obtained with low femtomole amounts applied to the MALDI target plate and for peptides such as angiotensin I and II with application of attomole amounts. Single scan acquisitions produce sufficient ion current even from proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), along with electrospray ionization (ESI), has had an enormous impact on science because of the capability to ionize nonvolatile and high-mass compounds such as peptides and * To whom correspondence should be addressed. † Wayne State University. ‡ The University of the Sciences in Philadelphia. 10.1021/ac902066s CCC: $40.75  2010 American Chemical Society Published on Web 11/11/2009

proteins.1,2 MALDI is characterized by the production of lowcharge state ions with singly charged ions being dominant, especially for compounds with molecular weights below 10-20 kDa while ESI3 is characterized by much higher charge states sometimes reaching hundreds of charges per molecule for larger proteins and polymers. The observation of mostly singly charged ions in MALDI reduces mass spectral complexity, which is especially useful for mixtures such as those found in polymer distributions, but they limit structural characterization because of the difficulty of producing fragment ions and requires a mass analyzer with a mass-to-charge (m/z) range at least equal to the molecular weight of the compounds being analyzed. Here, we report the ability to selectively produce either ESIlike multiply charged or MALDI-like singly charged ions using a MALDI process at atmospheric pressure (AP). Thus, multiply charged molecular ions dominate the mass spectrum when the analyte along with the common MALDI matrix, 2,5-dihydroxybenzoic acid (2,5-DHB), is applied to a glass MALDI target plate using the standard dried droplet sample preparation method2 and ablated using a nitrogen laser (337 nm). Under the same instrumental conditions, except that the sample preparation is by the solvent-free method4,5 where analyte and matrix are ground together and placed on the MALDI target plate without the use of any solvent, MH+ singly charged molecular ions are dominant. The ability to produce, at will, multiply charged ions in a MALDI process is potentially analytically important as it extends the mass range of common atmospheric pressure (1) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151–153. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299–2301. (3) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4671–4675. (4) Trimpin, S.; Rouhanipour, A.; Az, R.; Ra¨der, H. J.; Mu ¨llen, K. Rapid Commun. Mass Spectrom. 2001, 15, 1364–1373. (5) Trimpin, S.; McEwen, C. N. J. Am. Soc. Mass Spectrom. 2007, 18, 377– 381.

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Scheme 1. Representation of FF-TG AP-MALDI Source Design

ionization (API) mass spectrometers and provides enhanced user initiated fragmentation, similar to ESI. AP-MALDI offers advantages over vacuum MALDI, including time and volatility constraints as well as the ability to analyze materials under more physiologically relevant conditions, which is especially important in the developing area of compound-specific tissue imaging. Vacuum MALDI which is more sensitive than APMALDI, and often more sensitive than ESI,6 imposes analysis limitations with materials that are not compatible with vacuum and solvent conditions.7 Thus, methods that offer improved sensitivity for AP-MALDI are needed. Some sensitivity improvement for AP-MALDI is achieved by the implementation of pulse dynamic focusing (PDF),8 but part of the sensitivity gain is due to sampling a larger area. Thus, PDF only provides minimal sensitivity gain for imaging where spatial resolution (small sampled area) is important. A new AP-MALDI configuration based on theoretical studies by Sheehan and Willoughby9 and by laser ablation studies by Willis and Grosu10 is reported here. Sheehan and Willoughby modeled AP ionization and reported that >99% of the ions entering the AP to vacuum orifice are lost to the walls (so-called rim losses) if a high electric field is present in the AP region.9 Forward momentum caused by the explosive expansion of materials irradiated in a transmission geometry (TG) laser ablation experiment in which the laser beam passes through the holder (glass or quartz) before striking the sample was demonstrated by Willis and Grosu.10 Thus, combining these ideas into the configuration shown in Scheme 1 with field-free (FF) conditions to minimize rim loses and placement of the sample in close proximity to the ion entrance orifice to enhance momentum transfer of material in the expanding gas jet is expected to ensure high transmission of ions into the mass analyzer. TG using glass microscope slides as the MALDI target plate is also ideal for such a source configuration as well as for tissue imaging microscopy.11 The (6) Vestal, M. L. J. Mass Spectrom. 2009, 44, 303–317. (7) Trimpin, S.; Brizzard, B. Biotechniques 2009, 46, 409–419. (8) Clarke, S. L.; Vasanthakumar, A.; Anderson, S. A.; Pondarre´, C.; Koh, C. M.; Deck, K. M.; Pitula, J. S.; Epstein, C. J.; Fleming, M. D.; Eisenstein, R. S. EMBO J. 2006, 25, 544–553. (9) Sheehan, E. W.; Willoughby, R. C. U.S. Patent 7,060,976; June 13, 2006. (10) Willis, D. A.; Grosu, V. Appl. Phys. Lett. 2005, 84, 244103. (11) Navratil, M.; Mabbott, G. A.; Arriaga, E. A. Anal. Chem. 2006, 78, 4005– 4019.

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initial results from this ion source, with minimal optimization, demonstrate the FF-TG AP-MALDI concept by successfully obtaining mass spectra of lipids, carbohydrates, peptides, proteins, and polymers. The potential analytical utility of this new MALDI approach is discussed and representative examples presented. EXPERIMENTAL SECTION In the experiments discussed here, the laser (Spectra Physics, VSL-337ND-S nitrogen laser) is aligned so that the laser beam enters the ion entrance capillary of the ThermoFisher (Bremen, Germany) Orbitrap Exactive at approximately 180°. The beam is focused to a spot size of less than 100 µm at about 1 mm distance from the ion entrance orifice (Scheme 1). The matrix/analyte sample is applied to a glass microscope slide (Gold Seal, Portsmouth, NH, 80% UV transmission) and placed so that the sample faces the ion entrance orifice at about 1 to 2 mm distance from the orifice. Matrixes, sugars, and proteins were acquired from Sigma Aldrich (St. Louis, MO), peptides were from American Peptide Company (Sunnyvale, CA), lipids from Cayman Chemicals (Ann Arbor, MI), and polymers from Polymer Standards Services (Mainz, Germany). For solvent-based matrix/analyte preparation, the dried droplet method2 was used in which both matrix and analyte are dissolved in a common solvent, acetonitrile/water (ACN/H2O), either 70:30 for lipids or 50:50 for peptides/ proteins, and 1 µL of the solution placed on the microscope slide and dried using a heat gun (low heat). The faster evaporation due to the applied low heat (heat gun) insures a more even matrix/analyte surface and speeds up the sample preparation process but is not required for obtaining good results. The samples are prepared in matrix/analyte ratios (200/1 to 5000/1) similar to standard MALDI preparation methods.2 Alternatively, the solvent-free sample preparation method was used as described elsewhere.4,5 In order to observe ions in a TG experiment, the laser fluence must be sufficiently high to produce an explosive expansion of matrix/analyte that penetrates the matrix layer. The fluence is substantially higher than that used with most MALDI experiments (typically on the order of 1 J/cm2). The higher laser fluence does not result in detrimental effects on the observed mass spectra, including resolution, in AP-MALDI. All data were acquired with the Ion Max source removed and without either external voltages or external gas flow by overriding the interlocks. The capillary voltage (typically 20-140 V) was used with the electrospray ionization tune condition as was the tube lens voltage (typically 80-150 V). Caution needs to be exercised to ensure that there is no potential for exposure to the laser beam or hot surfaces near the ion entrance orifice. Mass spectra were also successfully acquired with the ion source housing in place by removing the glass window at the front of the source housing (Scheme 1). The ion transfer capillary was heated to 350 °C. Resolution could be set from 10 000 to 100 000 (m/z 200, full width at half height definition) and the maximum ion inject time was set to 50 ms. RESULTS AND DISCUSSION The mass spectra obtained from laser ablation of the solution based matrix/analyte preparation dried on a glass microscope slide using FF-TG AP-MALDI are nearly identical to ESI mass

Figure 1. FF-TG AP-MALDI inset mass spectra showing multiply charged molecular ions for (A) bovine insulin oxidized B chain (MW 3536.7, +3), (B) amyloid (1-42) (MW 4511.5, +4), (C) bovine pancreas insulin (MW 5772.6, +5), (D) PEG 6690 (+6), (E) ubiquitin (MW 8561, +7), and (F) cytochrome c (MW 12 224, +12).

spectra. Thus, singly charged ions are produced from compounds that do not have basic sites for attachment of more than one proton. Therefore, only singly charged ions are observed with FFTG AP-MALDI using solvent-based sample preparation with 2,5DHB, as well as with ESI using 50:50 acetonitrile/water, for lipids such as N-arachidonoylethanolamine (molecular weight [MW] 347.3), N-arachidonoyl glycine (MW 361.3), phospatidyl glycerol (MW 770.5), sphingomyelin (MW 812.7), and phospatidyl inositol (MW 886.6) as well as small peptides such as leucine enkephalin (MW 555.3) and β-amyloid 33-42 (MW 914.5). However, for bradykinin antagonist (MW 1109.6), angiotensin I (MW 1295.7), angiotensin II (MW 1045.5), and adrenocorticotropic hormone (ACTH 18-39 human; MW 2464.2) using the same solvent-based sample preparation conditions with AP-MALDI or ESI, multiply charged ions dominated the mass spectrum while singly charged ions were either not observed or in very low abundance. Under these FF-TG AP-MALDI conditions, higher mass peptides, proteins, and synthetic polymers produce higher charge states: insulin B chain oxidized, (MW 3536.7) < β-amyloid (1-42), (MW 4511.5) < porcine insulin (pancreas, MW 5772.6) < polyethylene glycol, (PEG) 6590 < ubiquitin, (bovine red blood cells, MW 8561)

(Figure 1A-F). Ubiquitin for example displayed ions from charge state +5 to +13. The mass spectrum of cytochrome c (MW 12 224) obtained from a single 1 s acquisition of 4 pmol applied to a microscope slide shows multiply charged ions from +8 to +15 in Figure 2A. As with ESI, the higher charge states allow MALDI mass spectra to be obtained for compounds beyond the mass limitation (m/z 4000) of the mass spectrometer. Mass spectra can be obtained from complex mixtures such a protein digests or polymers. Multiply or singly charged mass spectra were obtained from narrow polydisperse PEG samples with MWs below the 4000 m/z limit of the mass spectrometer using 2,5-DHB and a trace of NaCl employing solvent-based or solvent-free preparation, respectively. However, molecular ions of PEG 6590 were only observed with the solvent-based sample preparation as multiply charged ions that fall within the m/z range of the mass spectrometer (Figure 2B). Ionization with the singly or multiply charged mechanism was by Na+ adduction, showing that metal adduction is favored for this polymer when sodium salt is present. The ability to produce multiply charged ions in a MALDI process has important advantages. It potentially enables producAnalytical Chemistry, Vol. 82, No. 1, January 1, 2010

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Figure 2. Full-range TG AP-MALDI mass spectra: (A) 4 pmol of cytochrome c applied to the microscope slide with 1 s acquisition at 100 000 resolution (MW 12 224) and (B) PEG 6690 in 2,5-DHB and NaCl prepared solvent-free and then dissolved in 70:30 ACN/H2O and 1 µL placed on the microscope slide using the dried droplet method.

tion of enhanced fragmentation from processes such as collisionally induced dissociation (CID),12 electron transfer dissociation (ETD),13,14 and electron capture dissociation (ECD).15 It also extends the mass range of mass spectrometers with limited m/z range, typically found with AP ionization instruments. Additionally, just as with nanospray ESI, the amount of sample necessary for an analysis can be reduced by minimizing the amount of matrix/ analyte solution placed on the glass slide. Sufficient signal was obtained in a single laser shot to produce the isotope distribution of the doubly charged ions from 0.1 µL of a 400 amol solution (80 amol) of angiotensin II in 2,5-DHB placed on a glass slide. Singly charged molecular ions can be produced for the compounds discussed above that are within the m/z limit of the Orbitrap Exactive simply using the solvent-free sample preparation method with 2,5-DHB. The advantage of producing only singly charged ions is the simplified mass spectra obtained from complex mixtures such as found in plasma or even synthetic polymers. The mechanism for producing singly charged ions is distinct from (12) Trimpin, S.; Mixon, A. E.; Stapels, M. D.; Kim, M. Y.; Spencer, P. S.; Deinzer, M. L. Biochemistry 2004, 43, 2091–2105. (13) Syka, J.; Coon, J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9528–9533. (14) Gunawardena, H. P.; Emory, J. F.; McLuckey, S. A. Anal. Chem. 2006, 78, 3788–3793. (15) Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. A.; Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. Anal. Chem. 2000, 72, 563–573.

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Figure 3. FF-TG AP-MALDI mass spectrum of angiotensin I: (A) solvent-based dried droplet sample preparation using 2,5-DHB in 50: 50 ACN/H2O and (B) solvent-free sample preparation using 2,5-DHB.

that for producing multiply charged ions and is shown to originate in the matrix choice or matrix/analyte preparation method. This is shown in Figure 3 for angiotensin I prepared using 2,5-DHB employing the solvent-based (Figure 3A) and solvent-free (Figure 3B) methods. The mass spectra obtained with the singly charged mechanism often displayed matrix or Na+ adduction, especially for peptides. These adductions were rarely observed and then in low abundance with the multiply charged ionization mechanism. It was even possible with 2,5-DHB to produce a mixed mode of ionization with observation of singly and multiply charged ions by, for example, adding a drop of solvent onto the solvent-free prepared matrix analyte mixture on the glass plate. After solvent evaporation, the matrix/analyte mixture is only partially dissolved and the mixed mode ionization can be observed. The activation of the singly charged ion mechanism was also achieved using a thin layer experiment and increasingly nonincorporating conditions. Laser ablation of an aqueous solution of 2,5-DHB placed on angiotensin II which had been dried on the glass slide produced, after drying, exclusively doubly charged ions whereas an acetone solution of 2,5-DHB placed over the dried peptide produced mixed ionization and a dichloromethane 2,5-DHB solution produced only singly charged ions. With CHCA, even under solvent-based conditions, only singly charged ions are observed for angiotensin II and substance P. This observation is in line with previously described field-

constrained TG AP-MALDI experiments.16 Other compounds such as dithranol, ortho-chlorobenzoic acid, and benzoic acid were examined as matrixes for AP-MALDI. With angiotensin II, benzoic acid produced singly and no doubly charged ions while odichlorobenzoic acid produced low abundance doubly charged ions. Dithranol, besides producing singly charged ions with angiotensin II, also produced notable matrix-adducted analyte molecular ions. Previous studies have shown a moderate increase in multiple charging in MALDI using different sample preparations, high laser fluence, a metal-free sample stage, and atmospheric pressure.17,18 From the work presented here, it is clear that the MALDI process can generate analytically useful ion currents from mechanisms that produce multiply charged ions as well as singly charged ions. The ease and speed of the FF-TG AP-MALDI method for producing either singly or multiply charged ions from a wide variety of compound types with high sensitivity using a focused laser beam demonstrates analytical utility. Additional mass spectra obtained using the FF-TG AP MALDI method are displayed in the Supporting Information. CONCLUSIONS A unique new laser-based ionization method for production of multiply charged ions having good sensitivity, without optimization, at high laser spatial resolution (