Anal. Chem. 2010, 82, 4413–4419
Matrix-Enhanced Secondary Ion Mass Spectrometry (ME SIMS) Using Room Temperature Ionic Liquid Matrices Jennifer J. D. Fitzgerald, Paul Kunnath, and Amy V. Walker* Department of Materials Science and Engineering, University of Texas at Dallas, 800 West Campbell Road, RL 10, Richardson, Texas 75080 Room temperature ionic liquids (ILs) have many applications including as matrices in MALDI. We wished to investigate the efficacy of ILs as matrices in time-of-flight secondary ion mass spectrometry and in mass spectrometric imaging (MS imaging). Two ILs derived from r-cyano-4-hydroxycinnamic acid (CHCA) were synthesized and tested using phospholipids, cholesterol, and peptides. The molecular ion intensities of 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-snglycero-3-phosphoethanolamine (DPPE), cholesterol, and bradykinin were greatly increased using IL matrices. Further, detection limits were also improved: for DPPC and DPPE detection, limits were at least 2 orders of magnitude better using IL matrices. However, these IL matrices were not effective for the enhancement of angiotensin I ions. The data also indicate that IL matrices are suitable for imaging MS. The IL matrices did not cause changes to the sample surface via matrix crystallization or other processes; no “hot spots” were observed in the mass spectra. As a demonstration, an onionskin membrane was imaged. In the matrix-enhanced MS images, ions characteristic of proteins and other biomolecules were observed which could not otherwise be observed. Clearly ionic liquids deserve further investigation in SIMS and MS imaging. Imaging mass spectrometry (MS) provides a unique opportunity to obtain insights into a wide range of samples from biological tissues to molecular electronics. In this method, the spatial distribution of atoms and molecules, including polymers, pharmaceuticals, lipids, proteins, and semiconductors, is acquired without the use of chemical labels such as fluorescent tags.1-8 Currently, two ionization techniques are used to record MS images: matrix-assisted laser desorption ionization (MALDI)1-4,7,8 and secondary ion mass spectrometry (SIMS).1,5-7 MALDI is often employed to image biological samples because it can easily ionize large nonvolatile biomolecules, such as proteins and nucleotides.4 In imaging MALDI, the image is obtained by scanning the sample through the laser beam.1,7 The spatial resolution is determined by the laser spot size, typically ∼25 µm,3,7,8 which is insufficient to obtain the spatial distribution of * Corresponding author: (e-mail)
[email protected]; (phone) 972 883 5780; (fax) 972 883 5725. 10.1021/ac100133c 2010 American Chemical Society Published on Web 05/12/2010
molecules within a cell or nanoscale object. One disadvantage of MALDI imaging is that the sample must be carefully prepared to maintain the spatial arrangement of the compounds present.9 SIMS has some advantages over MALDI for imaging. The lateral resolution is better, and can be as high as 200 nm.6 It also does not require the careful application of a matrix. In SIMS, a high energy primary ion beam impinges on the sample surface leading to the ejection of secondary speciessneutrals, electrons, and ions.10 Imaging is usually performed in a microprobe configuration in which a focused primary ion beam is rastered across the sample surface.5,10 In this configuration, the lateral resolution of the image is determined by the analyte ionization probability and concentration, and the ion beam diameter as well as other instrument characteristics.10 One of the major issues in SIMS is that the ionization probability of intact molecules with m/z > 500 is extremely low leading to lateral image resolutions that are much lower than the ion beam diameter.5,11 For example, for 25 keV Ga+ primary ions, the beam diameter is 100 nm but the lateral resolution .1 µm.11 Cluster ion beams, including Aun+,11,12 Binx+,11,13-15 and C60x+,11,16,17 greatly improve the efficiency of secondary ion formation from molecules particularly at high mass. (1) McDonnell, L. A.; Heeren, R. M. A. Mass Spectrom. Rev. 2007, 26, 606– 643. (2) Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69, 4751–4760. (3) Stoeckli, M.; Chaurand, P.; Hallahan, D. E.; Caprioli, R. M. Nat. Med. 2001, 7, 493–496. (4) Todd, P. J.; Schaaff, T. G.; Chaurand, P.; Caprioli, R. M. J. Mass Spectrom. 2001, 36, 355–369. (5) Pacholski, M. L.; Winograd, N. Chem. Rev. 1999, 99, 2977–3005. (6) Walker, A. V. Anal. Chem. 2008, 80, 8865–8870. (7) Rubakhin, S. S.; Jurchen, J. C.; Monroe, E. B.; Sweedler, J. V. Drug Discovery Today 2005, 10, 823–837. (8) Chaurand, P.; Schwartz, S. A.; Caprioli, R. M. Curr. Opin. Chem. Biol. 2002, 6, 676–681. (9) Schwartz, S. A.; Reyzer, M. L.; Caprioli, R. M. J. Mass Spectrom. 2003, 38, 699–708. (10) Walker, A. V. In The Encyclopedia of Mass Spectrometry: Molecular Ionization Methods; Gross, M. L., Caprioli, R. M., Eds.; Elsevier Science & Technology Books: Burlington, VT, 2007; Vol. 6, p 535-551. (11) Kollmer, F. Appl. Surf. Sci. 2004, 231-232, 153–158. (12) Nagy, G.; Gelb, L. D.; Walker, A. V. J. Am. Soc. Mass Spectrom. 2005, 16, 733–742. (13) Touboul, D.; Kollmer, F.; Niehuis, E.; Brunelle, A.; Lapre´vote, O. J. Am. Soc. Mass Spectrom. 2005, 16, 1608–1618. (14) Nagy, G.; Walker, A. V. Int. J. Mass Spectrom. 2007, 262, 144–153. (15) Winograd, N. Anal. Chem. 2005, 77, 143A–149A. (16) Wong, S. C. C.; Hill, R.; Blenkinsopp, P.; Lockyer, N. P.; Weibel, D. E.; Vickerman, J. C. Appl. Surf. Sci. 2003, 203-204, 219–222. (17) Carado, A.; Kozole, J.; Passarelli, M.; Winograd, N.; Loboda, A.; Bunch, J.; Wingate, J.; Hankin, J.; Murphy, R. Appl. Surf. Sci. 2008, 255, 1572–1575.
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This has led to images with improved contrast and increases the experimental lateral resolution from molecular ions to ∼400 nm.13 However, this lateral resolution is still much larger that the diameter of the primary ion beam, ∼100 nm.11 A second method by which to enhance secondary ion intensities (by increasing the ionization probability of the analyte) is to employ sample pretreatment methods. The combination of sample pretreatment methods with a cluster ion beam source has the potential to greatly improve the capabilities of imaging SIMS. In matrix-enhanced SIMS (ME SIMS), a matrix, such as 2,5 dihydroxybenzoic acid (2,5 DHB), glycerol, or hydrochloric acid, is mixed with, or sprayed onto, the analyte.18-23 Using this method, quasimolecular ions from proteins up to m/z 10 000 have been detected.18 However, ME SIMS is unsuitable for use in imaging applications because the application of a matrix can lead to changes in the sample surface due to crystallization of the applied matrix.18,22,23 This causes the recorded distribution of the atoms and molecules present on the surface to be different from the original (“true”) distribution. This phenomenon is often manifested via the formation of “hot spots”, which are areas where the recorded ion intensities are much larger than elsewhere on the sample surface. The addition of metals, such as gold and silver, to a sample can also increase the intensity of secondary ions detected (meta-SIMS).24-26 Although meta-SIMS is suitable for imaging, it can be difficult to assign peaks and to interpret the images obtained.1,27 Further, Adriaensen et al.27 have reported that meta-SIMS ion yields are time dependent and do not correspond to the concentration of the analyte. Recently, room temperature ionic liquids (ILs) have been shown to be effective MALDI matrices. They can be employed with a wide variety of analytes including polymers,28,29 oligonucleotides,30,31 peptides,28,31 proteins,28,31 lipids,32 oligosaccarides,29 and glycoconjugates.29 In MALDI, the performance of IL matrices has been demonstrated to be at least as good as, if not better than, the analogous solid matrix. For example, using a solid R-cyano4-hydroxycinnamic acid (CHCA) matrix, the detection limit of bradykinin was 10 pmol/mL (10-5 mg/mL).28 Using an IL matrix, the detection limit was more than 3 orders of magnitude better,
