Researchers in a number of areas, from agriculture to environmental processes to pharmacology, want to know more about the structure, function and biosynthetic pathways of metabolites in plants. Because of the number and diversity of metabolites — there are an estimated 200,000 in the plant kingdom -- they have had to develop a range of methods to achieve the selectivity and sensitivity needed for analysis in complex mixtures. Methods have included those based on mass spectrometry, on nuclear magnetic resonance and on vibrational spectroscopy.Many researchers have come to rely on mass spectrometry in particular, because it offers inherently high sensitivity and specificity. However, many of the techniques currently used involve sample preparation steps that can inhibit the study of live biological samples. Investigators with George Washington University in Washington therefore have reported an atmospheric pressure infrared Maldi imaging mass spectrometry technique for plant metabolomics. As described in the Jan. 15 issue of Analytical Chemistry, the technique overcomes the various barriers to probing live biological samples that they might have faced with other similar methods.Infrared MaldiThe researchers achieved this through the combination of infrared and atmospheric pressure Maldi. They noted that infrared Maldi has not caught on generally because it is not as analytically robust as conventional UV Maldi and because of the relatively high cost of mid-IR sources. However, it has advantages for particular applications. First, the technique has more potential matrixes because many molecules offer strong absorption in the mid-infrared. Also, investigators can couple it directly with some liquid-phase separation techniques.Similarly, although keeping the sample at atmospheric pressure can result in slightly lower sensitivity with respect to transferring it to the vacuum system of the mass spectrometer, it can be favorable in some cases. Because of the reduced sample handling involved with the technique and the applicability of a broader range of matrixes, the researchers wrote, atmospheric pressure Maldi could contribute to in vivo studies, among others.Akos Vertes, the principal investigator of the study, explained that research groups have experimented with infrared excitation and atmospheric pressure sources since the early 1990s and since early in this decade, respectively. “A few years ago, people started putting two and two together and combining mid-IR and atmospheric pressure techniques.” However, the researchers probed dried samples in these studies; they did not look at biological tissues directly. One of the two new components of the Analytical Chemistry paper, Vertes said, was the application of atmospheric pressure infrared Maldi mass spectrometry for investigations of biological tissues.The other was refinement of the technique for imaging. Early last year, the researchers described a study in which they achieved atmospheric pressure infrared Maldi imaging using native water in plant tissue as a matrix for positive ion production with infrared laser irradiation: Thus, they showed they could map the spatial distributions of surface peptides and various small molecules in the tissue without resorting to any external matrixes. In the current study, they reported experiments with a wide range of plant tissue types in both positive and negative ion modes as well as imaging of fluid transport resulting from plant transpiration.They used a homebuilt atmospheric pressure Maldi ion source with a 30-mm inlet capillary mounted on a Q-TOF mass spectrometer made by Waters Co. of Milford, Mass. Excitation was provided by a 10-Hz-repetition-rate Nd:YAG laser, the output of which was converted to 4-ns mid-infrared (2940-nm) pulses by an optical parametric oscillator. Beam steering was achieved using gold mirrors and beam focusing with a 50-mm-focal-length planoconvex lens. To achieve maximal ion signal intensity while avoiding an electrical breakdown, the researchers maintained a distance of approximately 2 mm between the mass spectrometer inlet orifice and the sample. They collected positive and negative ion spectra for all samples using the atmospheric pressure interface.Using this setup, they identified more than 50 small metabolites and various lipids, including, for instance, 70 percent of the intermediates in the citric acid cycle. Thus, they demonstrated the potential of the technique for quick identification of a wide range of metabolites. The technique recommends itself especially for analysis of living organisms because it enables analysis at atmospheric pressure and does not involve extensive sample preparation.The researchers noted, however, that there still is room for improvement. Demonstration of reliable quantitation of metabolites in tissue samples would advance the technique considerably. Investigators have reported absolute quantitation of a range of metabolites using gas chromatography mass spectrometry. Comparative studies with atmospheric pressure infrared mass spectrometry could help to establish these numbers for the latter technique.Researchers have reported an atmospheric pressure infrared Maldi imaging mass spectrometry technique for plant metabolomics that offers a variety of advantages over others available for the same purpose. It allows quick identification and imaging of metabolites in live samples, for example. Also, it enables ablation of subsurface layers, facilitating depth profiling and possibly 3-D imaging of samples. Shown here are scanning electron microscopy images of infrared laser ablation of a cilantro leaf, after one laser shot (top) and five laser shots (bottom).In addition, the technique would benefit from improvements in instrumentation. Better focusing would result in improved spatial resolution, for example. At the same time, higher repetition rates would facilitate more reasonable collection times. The investigators would like to move from the 10 to 20 Hz of the current setup to the kilohertz scale. No such lasers are available, so they are working with a company to develop a 100-Hz laser.Finally, they hope to explore further the technique’s potential for depth profiling. “If you expose the same spot on the sample to multiple laser shots, you can actually dig into the sample and ablate material from subsurface layers, resulting in depth profiling,” Vertes explained. To exploit this possibility, they first will need to establish the removal rates of tissues through laser ablation. The payoffs could be considerable, however. “If you combine depth profiling with lateral imaging, you get 3-D imaging,” Vertes said, opening up a range of additional applications.