Correspondence/Rebuttal pubs.acs.org/est
Response to Comment on “MALDI-MS Imaging Analysis of Fungicide Residue Distributions on Wheat Leaf Surfaces”
W
e thank Dong et al. (2015)1 for their comments on our paper.2 Dong et al.,1 requested clarity regarding the (1) effect of variations in the leaf surface on MALDI-MS, (2) measurement of limits of detection, (3) correlation between droplet size of the spray and spatial resolution of the MALDI image, and (4) MALDI image resolution and quantitative aspects of the measurement. MALDI imaging of leaf surfaces to determine spatial distribution of endogenous and exogenous compounds has been very well documented in the literature.3−7 As Dong et al.1 pointed out, uneven surfaces can affect the mass accuracy of the data obtained.8 In our study,2 we ensured that the sample had a uniform matrix coating using an optical microscope as described by Araújo et al.9 in addition the sample was flattened to the extent that any surface height variations had a minimal effect on the mass accuracy of the measurement. To demonstrate the quality of our measurements, we overlaid 256 individual mass spectra obtained from MALDI imaging of a leaf applied with a 0.5 μL droplet containing pyraclostrobin as described in our paper.2 The overlaid MS spectra in Figure 1
probability for this process to contribute to banding effect, increasing the likelihood that the “banding effect” was a result of formulation spreading. The primary goal of our study was to establish a method to determine the spatial distribution of fungicide on leaf surface using MALDI-TOF imaging following track sprayer applications. Since plant leaves are randomly oriented on intact plants within a pot, it is impossible to obtain a uniform deposition of the sprayed fungicides on the leaf surface. Therefore, we have little control over spray retention on individual leaves which can account for variability in the detection of the fungicides. Furthermore, in Figures 4A and 5A of our study,2 we presented the MALDI images of the matrix background which can serve as a visual control to compare with the MALDI images of the fungicides (Figures 4B,C and 5B−D)2 and deduce any variability contributed by the matrix spraying process. In our study,2 it was necessary to determine the practical minimal amount (or detection limit) of applied fungicide, allowing us to consistently and reproducibly detect them across several imaging experiments. We did not intend to determine the absolute limits of detection (LOD), but rather aimed to obtain an optimal detection level to help us guide in designing follow-up experiments throughout our study. Although it was not indicated in our paper,2 detection at 0.03 μg or lower amount/spot did not reproducibly allow detection of the parent peak, while the 0.06 μg/spot of pyraclostrobin produced consistently an acceptable signal/noise of the parent peak under the same conditions. Therefore, this value should not be used as a truly quantitative LOD, and a true LOD measurement would require a more detailed study design. Furthermore, we would like to note that the variability introduced by the formulation and leaf texture may greatly influence the fungicide’s distribution, making it extremely challenging to obtain an accurate calibration curve to quantify the amount of fungicides across leaf surfaces. The TeeJet track sprayer nozzle used in our study generates droplets that are 144−235 μm in diameter,10,11 and the air brush used to coat the MALDI matrix on the leaf samples generates droplets which are typically 20−50 μm in diameter. Based on the droplet size generated by the track sprayer, we chose to use a spatial resolution of 300 × 300 μm to determine the fungicide distributions on the leaf surface. In summary, we thank Dong et al. for their constructive comments and the Editor for giving us an opportunity to clarify and present more data to reiterate the applicability of MALDI imaging method for industrial application to evaluate the distribution of xenobiotics on leaf surfaces.
Figure 1. Overlaid MS scans showing the protonated pyraclostrobin peak (m/z 388.1) generated in a MALDI-TOF imaging experiment. The mass shift of the pyraclostrobin peak shows change in sample thickness.
illustrates that the shift in the observed protonated pyraclostrobin peak (m/z 388.1) was within the mass window used to generate images in this study (±0.25%), and any observed mass shift could be attributed to the uneven surface of the leaf. The importance of choosing the appropriate mass window for generating the MALDI images was illustrated in Figure 2, wherein we show that using a smaller mass window (higher mass resolution) can skew the results due to variability associated with mass measurement under the experimental conditions which were used in these studies. The “banding effect” which was observed in some of the images may be an artifact of variation in matrix deposition caused by uneven surface of the leaf (before drying), or could be due to spreading of the fungicide formulation on the leaf surface after application. Since the authors used a finer nozzle for matrix application than for formulation application, there was less © 2015 American Chemical Society
Suresh P. Annangudi Kyung Myung* Cruz Avila Adame Andrew J. Bowling Published: August 12, 2015 10747
DOI: 10.1021/acs.est.5b03670 Environ. Sci. Technol. 2015, 49, 10747−10749
Environmental Science & Technology
Correspondence/Rebuttal
Figure 2. MALDI images of wheat leaf generated for pyraclostrobin with varying mass windows. The pyraclostrobin monoisotopic peak from the average MS scan (panel on the right side) of the imaging experiment was used to generate three different MALDI images with a mass window of 0.1% (A, B, and C) and one image with a mass window of 0.25% (D). The larger mass window (D) was chosen in our study to account for mass shifts due to change in surface topography. by matrix-assisted laser desorption−ionization mass spectrometry imaging: glucosinolates on Arabidopsis thaliana leaves. Plant J. 2015, 81, 961−972. (4) Vrkoslav, V.; Muck, A.; Cvacka, J.; Svatos, A. MALDI imaging of neutral cuticular lipids in insects and plants. J. Am. Soc. Mass Spectrom. 2010, 21, 220−231. (5) Holscher, D.; Shroff, R.; Knop, K.; Gottschaldt, M.; Crecelius, A.; Schneider, B.; Heckel, D. G.; Schubert, U. S.; Svatos, A. Matrix-free UV-laser desorption/ionization (LDI) mass spectrometric imaging at the single-cell level: distribution of secondary metabolites of Arabidopsis thaliana and Hypericum species. Plant J. 2009, 60, 907− 918. (6) Lopes, A. A.; Pina, E. S.; Silva, D. B.; Pereira, A. M.; da Silva, M. F.; Da Costa, F. B.; Lopes, N. P.; Pupo, M. T. A biosynthetic pathway of sesquiterpene lactones in Smallanthus sonchifolius and their localization in leaf tissues by MALDI imaging. Chem. Commun. 2013, 49, 9989−9991. (7) Cha, S.; Zhang, H.; Ilarslan, H. I.; Wurtele, E. S.; Brachova, L.; Nikolau, B. J.; Yeung, E. S. Direct profiling and imaging of plant metabolites in intact tissues by using colloidal graphite-assisted laser desorption ionization mass spectrometry. Plant J. 2008, 55, 348−360.
Mallika Dasari Jeffrey R. Gilbert
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Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, Indiana 46268, United States
AUTHOR INFORMATION
Corresponding Author
*Phone:1-317-337-7104 ; fax: 1-317-337-3205; e-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Dong, D.; Zheng, W.; Zhao, C. Comment on “MALDI-MS imaging analysis of fungicide residue distributions on wheat leaf surfaces”. Environ. Sci. Technol. 2015, DOI: 10.1021/acs.est.5b02513. (2) Annangudi, S. P.; Myung, K.; Avila Adame, C.; Gilbert, J. R. MALDI-MS imaging analysis of fungicide residue distributions on wheat leaf surfaces. Environ. Sci. Technol. 2015, 49, 5579−5583. (3) Shroff, R.; Schramm, K.; Jeschke, V.; Nemes, P.; Vertes, A.; Gershenzon, J.; Svatoš, A. Quantification of plant surface metabolites 10748
DOI: 10.1021/acs.est.5b03670 Environ. Sci. Technol. 2015, 49, 10747−10749
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(8) Garden, R. W.; Sweedler, J. V. Heterogeneity within MALDI samples as revealed by mass spectrometric imaging. Anal. Chem. 2000, 72, 30−36. (9) Araújo, P.; Ferreira, M. S.; de Oliveira, D. N.; Pereira, L.; Sawaya, A. C. H. F.; Catharino, R. R.; Mazzafera, P. Mass spectrometry imaging: an expeditious and powerful technique for fast in situ lignin assessment in Eucalyptus. Anal. Chem. 2014, 86, 3415−3419. (10) TeeJet Technologies Catalog 51A. http://www.teejet.com/ media/461473/technical_information.pdf. (11) Womac, A. R.; Maynard, R. A.; Kirk, I. W. Measurement variations in reference sprays for nozzle classification. T. ASABE 1999, 42, 609−616.
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DOI: 10.1021/acs.est.5b03670 Environ. Sci. Technol. 2015, 49, 10747−10749
