Anal. Chem. 2003, 75, 2333-2341
Effect of Local Matrix Crystal Variations in Matrix-Assisted Ionization Techniques for Mass Spectrometry Stefan L. Luxembourg, Liam A. McDonnell, Marc C. Duursma, Xinghua Guo, and Ron M. A. Heeren*
FOM Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands
Intense intact molecular ion signals have been obtained from phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol using matrix-enhanced secondary ion mass spectrometry (MESIMS). It was found that the high-mass (m/z >500) regions of the ME-SIMS spectra closely resembled those obtained using matrix-assisted laser desorption/ionization (MALDI). Using high spatial resolution SIMS, a detailed investigation of dried-droplet samples was performed. Based on the detected Na+ and 2,5-DHB matrix signal intensities, different crystal types were distinguished, in addition to different sizes of crystals. Spatially mapping the pseudomolecular and fragment ions of the phospholipids revealed that the nature of the pseudomolecular ions formed, as well as the ratio of intact molecular to fragment ion, was dependent on the type and surface composition of the crystal. The observed chemical bias effects due to crystal heterogeneity and the resulting variation in desorption/ionization efficiency will complicate the interpretation of data obtained from matrixassisted mass spectrometric (imaging) techniques and is an important factor in the “hot spot” phenomenon frequently encountered in MALDI experiments. In this respect, imaging SIMS was found to be a versatile tool to investigate the effects of the local physicochemical conditions on the detected molecular species. Chemical imaging mass spectrometry offers unique opportunities for obtaining more insight into numerous complex biochemical processes. Recording the distribution of elements and biomolecules in cells and tissues will help to address many issues in today’s cell biology and medicine; applications vary from membrane dynamics1 and drug delivery systems2,3 to altered protein distributions in diseased cells.4 Currently, two ionization techniques are used for recording chemical images of biological * Corresponding author: (e-mail)
[email protected]; (tel) +31-20-6081234; (fax) +31-20-6684106. (1) Cannon, D. M., Jr.; Pacholski, M. L.; Winograd, N.; Ewing, A. G. J. Am. Chem. Soc. 2000, 122, 603-610. (2) Belu, A. M.; Davies, M. C.; Newton, J. M.; Patel, N. Anal. Chem. 2000, 72, 5625-5638. (3) John, C. M.; Odom, R. W.; Salvati, L.; Annapragada, A.; Fu Lu, M. Y. Anal. Chem. 1995, 67, 3871-3878. (4) Stoeckli, M.; Chaurand, P.; Hallahan, D. E.; Caprioli, R. M. Nat. Med. 2001, 7, 493-496. 10.1021/ac026434p CCC: $25.00 Published on Web 04/12/2003
© 2003 American Chemical Society
samples, namely, matrix-assisted laser desorption/ionization (MALDI)5,6 and secondary ion mass spectrometry (SIMS).7 MALDI is one of the most versatile techniques for the ionization of large, nonvolatile biologically relevant molecules, such as peptides, proteins, and nucleotides.5,8 In the MALDI imaging approach, spatial information is obtained by scanning the sample through the (UV)laser beam. Over the last couple of years, a number of reports have appeared using scanning MALDI to image biological tissue.4,9 The spatial resolution achieved in these studies is determined by the laser spot size and was optimized to be ∼25 µm. While this is insufficient to obtain the spatial distribution of the molecules within a cell (the diameter of an average animal cell is ∼20 µm), it has allowed the spatial distribution of proteins in a tissue to be recorded. Recently, Spengler and Hubert10 reported the development of a novel confocal microscope/laser scanning MALDI instrument that enabled small peptides to be imaged with micrometer resolution (and inks with submicrometer resolution). However, it is still questionable whether large proteins can be desorbed and ionized from submicrometer-size laser spots without extensive fragmentation. In SIMS, a high-energy primary ion beam is used to irradiate a sample surface. Upon impact of the primary ions, neutrals, molecular ions, and atomic ions are liberated from the sample surface. The secondary ions are subsequently mass analyzed. By scanning the primary ion beam over the sample surface, information on its chemical composition is obtained. The achieved spatial resolution is determined by the primary ion beam spot size, which is typically a few hundred nanometers. Although the lateral resolution in SIMS is much better than in MALDI, the ion formation process leads to a higher degree of fragmentation of large molecules. Among other factors, the degree of fragmentation roughly scales with the density of primary ions and is one of the distinctions between the two regimes of operation in SIMS, dynamic and static SIMS. Dynamic SIMS uses a relatively intense primary ion beam to sputter the sample surface. This results in the removal of material at a rate of 1-10 nm/s and destruction of (5) Karas, M.; Bachmann, D.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53-68. (6) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151. (7) Benninghoven, A. Surf. Sci. 1994, 299/300, 246-260. (8) Zenobi, R.; Knochenmuss, R. Mass Spectrom. Rev. 1998, 17, 337-366. (9) Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69, 4751-4760. (10) Spengler, B.; Hubert, M. J. Am. Soc. Mass Spectrom. 2002, 13, 735-748.
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most molecular species in the sample.11 Dynamic (imaging) SIMS has proven to be a powerful tool for recording the distribution of elements (Na, Ca, K, Mg, etc.) present in cells as well as for the imaging of isotopic gradients.12,13 In static SIMS, a much less intense primary ion beam is used.11,14 The primary ion dose (
