Anal. Chem. 2010, 82, 9164–9168
New Paradigm in Ionization: Multiply Charged Ion Formation from a Solid Matrix without a Laser or Voltage Charles N. McEwen,*,† Vincent S. Pagnotti,† Ellen D. Inutan,‡ and Sarah Trimpin*,‡ Department of Chemistry and Biochemistry, University of the Sciences, Philadelphia, Pennsylvania 19104, United States, and Department of Chemistry, Wayne State University, Detroit, Michigan 48202, United States Laserspray ionization (LSI) is a new approach to producing multiply charged ions from solids on surfaces by laser ablation of matrixes commonly used in matrix-assisted laser desorption/ionization (MALDI). We show that the only necessity of the laser for producing multiply charged ions is to deliver particles or droplets of the matrix/analyte mixture to an ionization zone which is simply a heated inlet to the vacuum of the mass spectrometer. Several other methods for delivering sample are demonstrated to produce nearly equivalent results. One example shows the use of an air gun replacing the laser and producing mass spectra of proteins by shooting pellets into a metal plate which has matrix/analyte applied to the opposite side and near the ion entrance inlet to the mass spectrometer. Multiply charged ions of proteins are produced in the absence of any electric field or laser and with only the need of a heated ion entrance capillary or skimmer. The commonality of the matrix with MALDI and the mild conditions necessary for formation of ions brings into question the mechanism of formation of multiply charged ions and the importance of matrix structure in this process. The importance of producing multiply charged ions of proteins earned a share of the 2002 Nobel Prize in chemistry1,2 and, along with matrix-assisted laser desorption/ionization (MALDI),3,4 changed the face of mass spectrometry (MS) and even science. Numerous methods have since been introduced suitable for ionization of proteins based on producing ions from solvent droplets in an electric field.5-7 Some of the methods combine laser ablation8-11 or laser induced acoustic desorption (LIAD) with * To whom correspondence should be addressed. E-mail: c.mcewen@ usp.edu (C.N.M.);
[email protected] (S.T.). † University of the Sciences in Philadelphia. ‡ Wayne State University. (1) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64. (2) Fenn, J. B. Angew. Chem., Int. Ed. 2003, 42, 3871. (3) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y. Rapid Commun. Mass Spectrom. 1988, 2, 151. (4) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299. (5) Harris, G. A.; Nyadong, L.; Fernandez, F. M. Analyst 2008, 133, 1297. (6) Venter, A.; Nefliu, M.; Cooks, R. G. Trends Anal. Chem. 2008, 27, 284. (7) Van Berkel, G. J.; Pasilisi, S. P.; Ovchinnikovia, O. J. Mass Spectrom. 2008, 43, 1161. (8) Nemes, P.; Vertes, A. Anal. Chem. 2007, 79, 8098.
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electrospray ionization (ESI),12-14 but all of these methods that produce multiply charged ions of proteins or peptides almost certainly use the same mechanism for ionization as ESI. Recently, the MALDI and ESI methods seemed to be combined when multiply charged ions were produced by laserspray ionization (LSI) from a solid MALDI matrix at atmospheric pressure (AP) using laser ablation.15,16 The mechanism was believed to be formation of highly charged matrix/analyte particles or droplets by the explosive deposition of energy into the matrix by the laser beam with subsequent release of multiply charged ions after evaporation of the matrix as the charged droplets passed through a heated capillary between AP and the vacuum conditions of the mass spectrometer.16,17 The solid matrix was viewed to be the solvent, similar to ESI, and the final mechanism for ion formation was proposed to be the same as in ESI but occurring within the heated AP to vacuum transfer region of the mass spectrometer. The importance of heat on the formation and abundance of multiply charged LSI ions was previously shown for angiotensin I.16 Recently, the formation of ions from highly charged matrix/ analyte clusters inside a heated ion transfer capillary was discussed as a new paradigm for mass spectrometry.17 In that paper, we speculated that charges on the laser ablated matrix/analyte droplets might be produced inside the ion transfer capillary linking the atmospheric pressure (AP) region with the vacuum of the MS. Molten droplets were described as formed from the solid state of the matrix/analyte LSI sample and are then transferred into the mass spectrometer heated ion transfer capillary where the highly charged droplets are desolvated to produce multiply charged ions (9) Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2006, 17, 1712. (10) Sampson, J. S.; Muddiman, D. C. Rapid Commun. Mass Spectrom. 2009, 23, 1989. (11) Shiea, J.; Huang, M.-Z.; Hsu, C.-Y.; Lee, C.-H.; Yuan, I.; Beech, J. Rapid Commun. Mass Spectrom. 2005, 19, 3701. (12) Cheng, S.-C.; Cheng, T.-L.; Chang, H.-C.; Shiea, J. Anal. Chem. 2009, 81, 868. (13) Dixon, R. B.; Sampson, J. S.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2009, 20, 597. (14) Heron, S. R.; Wilson, R.; Shaffer, S. A.; Goodlett, D. R.; Cooper, J. M. Anal. Chem. 2010, 82, 3985. (15) Trimpin, S.; Inutan, E. D.; Herath, T. N.; McEwen, C. N. Anal. Chem. 2010, 82, 11. (16) Trimpin, S.; Inutan, E. D.; Herath, T. N.; McEwen, C. N. Mol. Cell. Proteomics 2010, 9, 362. (17) McEwen, C. N.; Trimpin, S. Int. J. Mass Spectrom. 2010, DOI: 10.1016/ j.ijms.2010.05.020. 10.1021/ac102339y 2010 American Chemical Society Published on Web 10/25/2010
by the ion evaporation18 or charge residue19 models proposed for ESI. Further, matrixes such as 2,5-DHAP have been shown to lower the thermal requirements for desolvation of the matrix/ analyte clusters.20 The primary source of droplet charging to produce the highly charged LSI ions is, however, unknown and was originally speculated to be related to processes involved with laser ablation of the matrix/analyte16 at AP as a logical extension of processes described for MALDI.16,21-25 Here, we demonstrate that the laser is not a requirement to sensitively produce multiply charged ions from a solid matrix. EXPERIMENTAL SECTION The mass spectrometers used in this study were the Thermo Fisher Scientific Orbitrap Exactive (Bremen, Germany) and the Waters SYNAPT G2 (Manchester, England). The Exactive was operated by removing the ion source enclosure and overriding the interlocks that allow the instrument to operate. The ion transfer tube supplied with the instrument was used in these studies, and the temperature was adjusted from the software between 50 and 450 °C, but for most studies was set to 325 °C. The SYNAPT G2 was operated in the nano-ESI mode with the skimmer set at 40 V and using a source temperature of 150 °C.26,27 Glass and metal heated transfer tubes of lengths from 2 to 10 cm were constructed by attaching these tubes to the cone with Sauereisen cement no. P1 (Sauereisen, Pittsburgh, PA). For the metal heated transfer tubes, a nichrome wire was coiled around the tube and covered with Sauereisen cement. The temperature of the metal tube could be raised to as much as 250 °C by applying a voltage across the wire. The chemicals and solvents used in this study were obtained from Sigma Aldrich (St. Louis, MO) and were used without further purification. 2,5-Dihydroxacetophenone (2,5-DHAP) was MALDI grade and 2,5-dihydroxybenzoic acid (2,5-DHB) was 98% and was used as received. The matrix solutions were prepared at 5 mg mL-1 or in the case of 2,5-DHAP as a saturated solution in 1:1 acetonitrile-water (HPLC grade). The 2,5-DHAP solution was warmed to ∼60 °C to increase the concentration of the solution. Typically, for these studies, the matrix solution was mixed in a 1:1 ratio with the analyte solution before deposition onto the target plate using the dried droplet method.4 For SYNAPT G2 acquisitions, 1 µL of the analyte was used, added with 1 µL of a matrix solution on top, and briefly mixed with the tip of the pipet. The concentration of 2,5-DHAP matrix was further increased by adding three 1 µL aliquots of matrix solution to the spotted droplet with mixing after each additions. In both methods the matrix/analyte spot was dried with warm air. Peptides and proteins were dissolved in water with the exception of bovine insulin which was first dissolved in a 1:1 methanol-water solution and then diluted in pure water. (18) Iribarne, J. V.; Thomson, B. A. J. Chem. Phys. 1976, 64, 2287. (19) Dole, M.; Mack, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. D.; Alice, M. B. J. Chem. Phys. 1968, 49, 2240. (20) McEwen, C. N.; Larsen, B. S.; Trimpin, S. Anal. Chem. 2010, 82, 4998. (21) Zhigilei, L. V.; Garrison, B. J. J. Appl. Phys. 2000, 88, 1281. (22) Schurenberg, M.; Schulz, T.; Dreisewerd, K.; Hillenkamp, F. Rapid Commun. Mass Spectrom. 1996, 10, 1873. (23) Knochenmuss, R. Analyst 2006, 131, 966. (24) Karas, M.; Kruger, R. Chem. Rev. 2003, 103, 427. (25) Karas, M.; Gluckmann, M.; Schafer, J. J. Mass Spectrom. 2000, 35, 1. (26) Inutan, E. D.; Trimpin, S. J. Am. Soc. Mass Spectrom. 2010, 21, 1260. (27) Inutan, E. D.; Trimpin, S. J. Proteome Res. 2010, DOI: 10.1021/pr10059.
The sample was also directly transferred to the instrument skimmer or ion transfer tube by gently tapping the area of dried matrix/analyte applied to a laboratory spatula, a melting point tube, or a glass microscope slide against the ion entrance aperture of the mass spectrometer. For the shockwave experiments of indirectly transferring the sample, an aluminum plate 3/16 in. thickness was mounted using a ring stand and held within 3 mm of the ion entrance aperture with the sample aligned with the ion entrance orifice. Blocks placed between the metal plate and the mass spectrometer on each side prevented its movement when force was applied. In one case, an air rifle BB gun (Daisey Pellet 0.177 calibar) using lead pellets was fired at the plate directly behind where the sample was mounted. For safety, a section of rubber tubing extended past the barrel and was pushed against the plate to catch the projectile and the operator wore a face shield. A Lisle (Lisle Corporation, Clarinda, IA) automatic center punch was also used to impart the shockwave in some studies by pushing the device against the plate opposite the sample until it automatically fired producing a shockwave. RESULTS AND DISCUSSION We previously reported that the temperature of the ion transfer capillary of the Thermo Fisher Scientific Ion Max source was crucial to forming abundant multiply charged ions of peptides with LSI, a process that produces ions by laser ablation of a matrix/ analyte mixture prepared identically to those successfully used in MALDI.16 We find that 2,5-DHAP as the matrix has a significantly lower optimum ion transfer capillary temperature than 2,5-DHB, but the multiply charged ion abundance for either matrix increase by at least 3 orders of magnitude from the onset to the optimum temperature. With either matrix, essentially no ions are observed with the capillary at ambient temperature. In contrast, singly charged ions produced by atmospheric pressure chemical ionization using the atmospheric solids analysis probe (ASAP) method28 are virtually unaffected by the temperature of the ion transfer capillary. Further, with ESI, under conditions of low solvent flow and high nebulization gas flow, where desolvation occurs before the ion transfer capillary, multiply charged ions are observed at ambient temperature and the abundance of the ions increase only slightly with increasing capillary temperature (Figure 1). Under higher solvent flow and low nebulization gas flow, about an order of magnitude increase in ion abundance for angiotensin I doubly charged ions was observed. Thus, if multiply charged ions had been produced by LSI before the ion transfer tube, they should be observed without application of heat. These results, therefore, conclusively demonstrated that few, if any, multiply charged ions are produced by LSI before the heated ion transfer capillary. Further, we also observed with LSI that after laser ablation the multiply charged ion signal decayed but did not immediately go to zero after firing the laser. This problem became noticeable in attempts at high-throughput analysis where extending the method below one second per sample analysis resulted in carryover of signal between adjacent samples. This observation led us to speculate that the ion transfer capillary might have a role in ion formation beyond desolvation of the charged droplets.17 Astonishingly, multiply charged ions are observed for peptides and proteins in the common MALDI matrixes 2,5-DHAP and 2,5(28) McEwen, C. N.; McKay, R. G.; Larsen, B. S. Anal. Chem. 2005, 77, 7826.
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Figure 1. The ion current chromatogram for the 1+, 2+, and 3+ charge state of angiotensin I by electrospray ionization on a Thermo Fisher Ion Max source at a flow rate of 10 µL min-1 and application of strong desolvation conditions with the ion transfer tube temperature increased from 100 to 450 °C. Little difference in ion abundance is observed as a function of the ion transfer temperature.
DHB by simply touching the matrix/analyte mixture on a spatula to the face of the heated ion transfer capillary of the Ion Max source. This capillary normally serves to transfer ions from AP to vacuum and provide additional desolvation when needed for ESI. Even with a capillary temperature of 150 °C, multiply charged ions are observed for insulin using 2,5-DHAP as matrix but as with LSI the ion current becomes more abundant at higher temperature. Ions are observed in the absence of either capillary or tube lens voltages, but a voltage (20 V) applied to the skimmer is necessary and is the same as used in ESI and APCI to guide ions through the intermediate pressure region between the ion transfer capillary and the lower pressure region of the ion optics of the mass analyzer. We refer to this new method of ionization as matrix assisted inlet ionization (MAII, pronounced “may I”). Figure 2 shows the summed mass spectrum obtained at optimum ion transfer capillary temperature from a mixture of ubiquitin and insulin at about 5 pmol loaded in 2,5-DHAP. The mass spectrum was obtained by application of the matrix/analyte mixture to the spatula using the dried droplet method and simply touching the mixture to the face of the ion transfer capillary. Similar results were obtained using a melting point tube for application of the sample. Further, ions from insulin were also observed using a similar procedure but with a Waters SYNAPT G2 mass spectrometer which uses a skimmer with a maximum temperature of 150 °C instead of an ion transfer capillary for the ion entrance from the AP to vacuum region. (see the supplemental figures in the Supporting Information). LSI, which uses laser ablation, is only one method for transferring the matrix/analyte to the ion transfer tube. We have not ruled out that charged droplets are formed by the laser beam impact on the matrix in LSI, but it is clearly not a prerequisite. To demonstrate that a shockwave is capable of producing mass spectra of multiply charged ions, a series of proteins and peptides 9166
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Figure 2. Mass spectrum of a mixture of ubiquitin and insulin in 2,5-DHAP matrix applied to a metal spatula using the dried droplet MALDI method and touched to the ion transfer tube entrance. The ion transfer tube was at 325 °C. Multiply charged ubiquitin ions from charge state 5+ to 11+ are observed and insulin ions from 3+ to 5+.
in 2,5-DHB or 2,5-DHAP were applied to a 3/16 in. thick plate of aluminum and a BB pellet was fired at high velocity at the back of the plate opposite the matrix/analyte sample. The plate was always positioned so that the sample was in front of and near the ion entrance orifice. After each pellet hits the aluminum plate, a signal is observed (data not shown). In a similar configuration, as little as 60 pmol of insulin in 2,5-DHAP applied to the plate produced a mass spectrum by simply pressing a Lisle automatic center punch device against the metal plate and on the opposite side to where the sample was applied. The ion transfer capillary was heated to 325 °C for these experiments. The center punch produces a shockwave somewhat more intense than the BB pellet. Only a single center punch shockwave was necessary to obtain the mass spectrum shown in Figure 3 for insulin. Even after several consecutive shockwaves, each generating a mass spectrum, no visual difference could be observed for the sample on the plate. Collecting dislodged matrix/analyte from a single center punch strike on a glass slide and examining it under a 20× microscope suggests that
