Anal. Chem. 1997, 69, 4751-4760
Molecular Imaging of Biological Samples: Localization of Peptides and Proteins Using MALDI-TOF MS
Richard M. Caprioli,* Terry B. Farmer, and Jocelyn Gile
Department of Biochemistry and Molecular Biology and Analytical Chemistry Center, University of Texas Medical School, 6431 Fannin Street, Houston, Texas 77030
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used to generate ion images of samples in one or more mass-to-charge (m/z) values, providing the capability of mapping specific molecules to two-dimensional coordinates of the original sample. The high sensitivity of the technique (lowfemtomole to attomole levels for proteins and peptides) allows the study of organized biochemical processes occurring in, for example, mammalian tissue sections. The mass spectrometer is used to determine the molecular weights of the molecules in the surface layers of the tissue. Molecules desorbed from the sample typically are singly protonated, giving an ion at (M + H)+, where M is the molecular mass. The procedure involves coating the tissue section, or a blotted imprint of the section, with a thin layer of energy-absorbing matrix and then analyzing the sample to produce an ordered array of mass spectra, each containing nominal m/z values typically covering a range of over 50 000 Da. Images can be displayed in individual m/z values as a selected ion image, which would localize individual compounds in the tissue, or as summed ion images. MALDI ion images of tissue sections can be obtained directly from tissue slices following preparative steps, and this is demonstrated for the mapping of insulin contained in an islet in a section of rat pancreas, hormone peptides in a small area of a section of rat pituitary, and a small protein bound to the membrane of human mucosa cells. Alternatively, imprints of the tissue can be analyzed by blotting the tissue sections on specially prepared targets containing an adsorbent material, e.g., C-18 coated resin beads. Peptides and small proteins bind to the C-18 and create a positive imprint of the tissue which can then be imaged by the mass spectrometer. This is demonstrated for the MALDI ion image analysis of regions of rat splenic pancreas and for an area of rat pituitary traversing the anterior, intermediate, and posterior regions where localized peptides were mapped. In a single spectrum from the anterior/ intermediate lobe of a rat pituitary print, over 50 ions corresponding to the peptides present in this tissue were S0003-2700(97)00888-3 CCC: $14.00
© 1997 American Chemical Society
observed as well as precursors, isoforms, and metabolic frag ments. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS)1 has been shown to be quite versatile in its many applications to the analysis of biological samples, especially to peptides and proteins. Typically, samples are mixed with an organic compound that acts as a matrix to facilitate ablation and ionization of compounds in the sample. Some recent work has shown that the application of thin layers of matrix has special advantages, particularly when very high sensitivity is needed.2-5 In earlier work, we described methods for preparation of cellulose membranes precoated with a thin matrix layer for the direct deposition and analysis of aqueous samples.2 This technique circumvents the problems of mixing and dilution of samples when postaddition of matrix is done and effectively allows small (nanoliter) volumes of samples to be applied to the target. Further, the method is an essential part of the development of an off-line capillary electrophoresis/MALDI MS analysis technique.6 Several reports have described the use of MALDI MS for the analysis of specific peptides in whole cells, e.g., for the analysis of some neuropeptides directly in single neurons of the mollusk Lymnaea stagnalis.7 Isolated neurons were ruptured, mixed with small volumes of matrix, and analyzed. In subsequent work, this group demonstrated the ability of MALDI MS to be used to elucidate some of the metabolic processing involved in neuropeptide production from precursor peptides.8 Also, a single neuron from Aplysia californica was analyzed for several specific neuropeptides by using a procedure involving removal of excess salt by rinsing with matrix solution.9 (1) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53-68. Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2301-2303. (2) Zhang, H.; Caprioli, R. J. Mass Spectrom. 1996, 31, 690-692. (3) Vorm, O.; Roepstorff, P.; Mann, M. Anal. Chem. 1994, 66, 3281-3287. (4) Axelsson, J.; Hoberg, A.-M.; Waterson, C.; Myatt, P.; Shield, G. L.; Varney, J.; Haddleton, D. M.; Derrick, P. Rapid Commun. Mass Spectrom. 1997, 11, 209-213. (5) Dwyer, J.; Botten, D. Am. Lab. 1996, (Nov), 51-54. (6) Zhang, H.; Caprioli, R. J. Mass Spectrom. 1996, 31, 1039-1046. (7) van Neelen, P. A.; Jime´nez, C. R.; Li, K. W.; Wildering, W. C.; Geraerts, W. P. M.; Tjaden, U. R.; van der Greef, J. Org. Mass Spectrom. 1993, 28, 15421546. (8) Jime´nez, C. R.; van Neelen, P. A.; Li, K. W.; Wildering, W. C.; Geraerts, W. P. M.; Tjaden, U. R.; van der Greef, J. J. Neurochem. 1994, 62, 404-407.
Analytical Chemistry, Vol. 69, No. 23, December 1, 1997 4751
The ability to image a sample for individual molecular compounds in order to obtain the detailed spatial arrangement of these compounds in an ordered target sample such as a slice of tissue could be of enormous value in biological research. Selected ion surface maps of such samples could provide details of compound compartmentalization, site-specific metabolic processing, and selective binding domains for a very wide variety of natural and synthetic compounds. Although no work thus far has been reported using MALDI for this purpose, a considerable amount of work has been described for use of secondary ion mass spectrometry (SIMS) for the spatial arrangement of elements in surfaces of samples including biological tissue and organic polymers.10 In addition, there have been recent efforts to apply SIMS to organic compounds and metabolites of low mass (
